Evolutionary and biogeographic patterns of the Badidae (Teleostei: Perciformes) inferred from mitochondrial and nuclear DNA sequence data

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 1010–1022 www.elsevier.com/locate/ympev

Evolutionary and biogeographic patterns of the Badidae (Teleostei: Perciformes) inferred from mitochondrial and nuclear DNA sequence data Lukas Ru¨ber a

a,*

, Ralf Britz b, Sven O. Kullander c, Rafael Zardoya

a

Departamento de Biodiversidad y Biologı´a Evolutiva, Museo Nacional de Ciencias Naturales, Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain b Division of Fishes, Smithsonian Institution, Washington, DC 20013-7012, USA c Department of Vertebrate Zoology, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, Sweden Received 17 December 2003; revised 20 April 2004 Available online 17 June 2004

Abstract We reconstructed phylogenetic relationships of the family Badidae using both mitochondrial and nuclear nucleotide sequence data to address badid systematics and to evaluate the role of vicariant speciation on their evolution and current distribution. Phylogenetic hypotheses were derived from complete cytochrome b (1140 base pairs) sequences of 33 individuals representing 13 badid species, and using three species of Nandidae as outgroups. Additionally, we sequenced the nuclear RAG1 (1473 base pairs) and Tmo-4C4 (511 base pairs) genes from each of the badid species and one representative of the outgroup. Our molecular data provide the first phylogenetic hypothesis of badid intrarelationships. Analysis of the mitochondrial and nuclear nucleotide sequence data sets resulted in well-supported trees, indicating a basal split between the genera Dario and Badis, and further supporting the division of the genus Badis into five species groups as suggested by a previous taxonomic revision of the Badidae. Within the genus Badis, mitochondrial and nuclear phylogenies differed in the relative position of B. kyar. We also used our molecular phylogeny to test a vicariant speciation hypothesis derived from geological evidence of large-scale changes in drainage patterns in the Miocene affecting the Irrawaddy– and Tsangpo–Brahmaputra drainages, in the southeastern Himalaya. Within both genera, Badis and Dario, we observed a divergence into Irrawaddy– and Tsangpo–Brahmaputra clades. Using a cytb substitution rate of 8.2 · 10 9 (substitutions · base pair 1 · year 1), we tentatively date this vicariant event at the Oligocene–Miocene boundary (19–24 Myr). It is concordant with a hypothesized paleo connection of the Tsangpo river with the Irrawaddy drainage that was most likely interrupted during Miocene orogenic events through tectonic uplifts in eastern Tibet. Our data, therefore, indicate a substantial role of vicariantbased speciation shaping the current distribution patterns of badids.
2004 Elsevier Inc. All rights reserved. Keywords: Phylogeny; RAG1; Tmo-4C4; Cytochrome b; Divergence times; Myanmar

1. Introduction The freshwater ichthyofauna of tropical Asia with around 2100 valid species, and an estimated total number of 3000 species is still poorly known (Lundberg et al., 2000). Recent research efforts that focused on new collections from previously un- or underexplored *

Corresponding author. Fax: +34 91 564 5078. E-mail address: [email protected] (L. Ru¨ber).

1055-7903/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.04.020

regions have added considerably to species numbers and towards a better understanding of the biodiversity of this highly endangered fauna (e.g., Kottelat, 2001). For example, until recently, the Indo-Burmese family Badidae (Teleostei, Perciformes) was considered to contain one single genus, and one to three species. New collections, and a taxonomic revision revealed that the family Badidae currently comprises 15 species, assigned to two genera Badis and Dario (Kullander and Britz, 2002).

L. Ru¨ber et al. / Molecular Phylogenetics and Evolution 32 (2004) 1010–1022

In the past, the genus Badis was generally treated as a member of the family Nandidae (leaffishes) together with the genera Nandus, Polycentrus, Monocirrhus, Afronandus, and Polycentropsis (the genus Pristolepis had also occasionally been included in the nandids). Nandids are usually classified as a Percoidei family, although close relationships to the Anabantoidei (labyrinth fishes) or the Channoidei (snakeheads) have been postulated (Gosline, 1968, 1971; Nelson, 1969; Rosen and Patterson, 1990). Based on morphological and behavioral data, a separate family, Badidae for Badis alone was erected by Barlow et al. (1968). Following Kullander and Britz (2002), the family Nandidae is now restricted to the genus Nandus and the genera Polycentrus, Monocirrhus, Afronandus, and Polycentropsis, formerly assigned to the Nandidae, are classified in the Polycentridae. Kullander and Britz (2002) hypothesized a sistergroup relationship of badids and nandids based on a uniquely shared derived character of the caudal skeleton. Kullander and Britz (2002) based on external morphological characters, but mostly on information derived from color patterns, assigned the Badis species to five species groups: B. assamensis species group (B. assamensis and B. blosyrus), B. badis species group (B. badis, B. kanabos, B. chittagongis, and B. ferrarisi), B. corycaeus species group (B. corycaeus and B. pyema), B. ruber species group (B. ruber, B. siamensis, and B. khwae), and B. kyar. In their revision of the Badidae, Kullander and Britz (2002) also erected a new genus Dario with three species (D. dario, D. hysginon, and D. dayingensis) for small badid fishes with adult size below 25 mm, which are morphologically clearly distinct from Badis. Nevertheless, the phylogenetic relationships among the badid species, and species groups still remain unresolved. Badids are small freshwater fish including the smallest percoid known so far (Dario dario with a standard length <20 mm). They normally inhabit small streams or hill streams with slow to moderate flow, coastal drainages or ditches with stagnant waters. They are lurking predators probably feeding on small invertebrates (Barlow et al., 1968). The distribution of the Badidae includes the Indian subcontinent, Pakistan, Nepal, Bangladesh, Myanmar, Peninsular Thailand, the Mae Khlong drainage, and part of the Mekong basin in South East Asia as well as the Upper Irrawaddy in southern Yunnan, China (Fig. 1, Table 1). The badid species are largely allopatric in distribution with few cases of sympatry: B. kanabos, B. blosyrus, and D. dario in western Assam, B. badis and B. assamensis in northern Assam, and B. kyar, B. corycaeus, and D. hysginon in northern Myanmar. Allopatric species with adjacent distribution suggest vicariant speciation as the main force underlying badid diversification (Kullander and Britz, 2002). It has long been recognized that paleo-drainages of major continental East Asian rivers, draining the southeastern Tibet plateau margin, differed markedly from

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Fig. 1. Map of India, Bangladesh, Myanmar, and adjacent regions showing the sampling localities of the Badidae used in this study. The numbers refer to the localities listed in Table 1. Major rivers mentioned in the text are indicated by circles filled in grey: B, Brahmaputra; G, Ganges; C, Chindwin; I, Irrawaddy; M, Mahanadi; and T, Tsangpo.

their current drainage patterns (Brookfield, 1998; Clark et al., 2004; Gregory, 1925; Gregory and Gregory, 1923; Hallet and Molnar, 2001; Me´tivier et al., 1999; Seeber and Gornitz, 1983; Zeitler et al., 2001). In a recent study, Clark et al. (2004) suggested that these rivers were once tributaries to a single southward flowing system, which drained into the South China Sea (Fig. 2A). Subsequent reorganization into modern major river drainages was primarily caused by river capture and reversal events associated with the initiation of Miocene uplifts in eastern Tibet (Clark et al., 2004). Although large-magnitude tectonic shear, prompted by the Indian–Asian collision around the eastern Himalayan syntaxis (especially in the ‘‘Three River’’ area where the Salween, Mekong, and Yangtze rivers run parallel, see Fig. 2), cannot be ruled out as an additional factor influencing these large-scale changes in drainage patterns (Clark et al., 2004; Hallet and Molnar, 2001). The evolution of drainage systems in Asia can be summarized in four stages (Fig. 2, Clark et al., 2004). (a) Upper Yangtze, Middle Yangtze, Upper Mekong, and Upper Salween rivers drained into the South China Sea through the paleo Red River (Fig. 2A). (b) Capture/reversal of the Middle Yangtze river redirected drainage away from the Red River and into the East China Sea through the Lower Yangtze river (Fig. 2B). (c) Capture of the Upper Yangtze river into the Lower Yangtze river, and of the Upper Mekong and Upper Salween rivers into their modern drainage position. The Tsangpo river was also captured to the south through the Irrawaddy river (Fig. 2C). (d) Capture of the Tsangpo river through the Brahmaputra river into its modern drainage position (Fig. 2D).

Species

Voucher

2cmID

GenBank Accession Nos. cytb

RAG1

Tmo-4C4

Badis Badis Badis

assamensis assamensis badis

NRM 41725 NRM 41718 NRM 41721

B261 B266 B259

AY330936 AY330937 AY330938

AY330966

AY330980

Badis

badis

NRM 48419

B041

AY330939

India, West Bengal, Calcutta (1)

Badis

badis

NRM 48420

B141

AY330939

India, West Bengal, Calcutta (1)

Badis

badis

NRM 41707

B268

AY330940

AY330967

AY330981

India, West Bengal, Tumapao river (2)

Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis Badis

blosyrus corycaeus corycaeus corycaeus corycaeus kanabos khwae khwae kyar kyar kyar kyar pyema pyema pyema pyema ruber

fin clip NRM 47929 NRM 41692 NRM 41689 NRM 41693 NRM 45101 CMK 17233 CMK 17233 NRM 41720 NRM 41695 NRM 41696 NRM 41694 NRM 47927 NRM 47928 NRM 47930 NRM 47931 fin clip

B284 B244 B272 B274 B276 B263 B286 B287 B264 B269 B270 B271 B042 B243 B245 B248 B040

AY330941 AY330942 AY330943 AY330944 AY330945 AY330946 AY330947 AY330947 AY330948 AY330948 AY330948 AY330949 AY330950 AY330950 AY330951 AY330950 AY330952

AY330968

AY330982

AY330969

AY330983

AY330970

AY330984

AY330971 AY330972

AY330985

India, Assam, Buxa Tiger reserve (3) Myanmar, Kachin State, Indawgyi lake (7) Myanmar, Kachin State, Myitkyina (6) Myanmar, Kachin State, Indawgyi lake (7) Myanmar, Kachin State, Myitkyina (6) India, Assam (Aquarium trade) Thailand, Kanchanaburi, Huai Ban Rai (9) Thailand, Kanchanaburi, Huai Ban Rai (9) Myanmar, Kachin State, Myitkyina (6) Myanmar, Kachin State, Myitkyina (6) Myanmar, Kachin State, Myitkyina (6) Myanmar, Kachin State, Myitkyina (6) Myanmar, Kachin State, Putao (5) Myanmar, Kachin State, Putao (5) Myanmar, Kachin State, Putao (5) Myanmar, Kachin State, Putao (5) unknown

Badis

ruber

NRM 41723

B260

AY330953

Badis Badis Badis Badis Dario

siamensis siamensis sp. ‘‘Assam’’ sp. ‘‘Assam’’ dario

NRM 48418 fin clip fin clip NRM 48421 NRM 45100

B034 B043 B014 B242 B262

AY330954 AY330955 AY330956 AY330957 AY330958

Dario Dario Dario Dario

hysginon hysginon hysginon hysginon

NRM NRM NRM NRM

47932 41691 41715 41690

B249 B265 B267 B273

fin clip NRM 48422 NRM 48423

B212 B033 B257

Outgroups Nandus nandus Nandus nebulosus Nandus oxyrhynchus

AY330986 AY330972 AY330973

AY330987

AY330974

AY330988

AY330975 AY330976

AY330989 AY330990

AY330977

AY330991

AY330959 AY330960 AY330961 AY330962

AY330978

AY330992

AY330963 AY330964 AY330965

AY330979

AY330993

Locality (number in Fig. 1)

Distribution

India, Assam, Dibrugarh area (4) India, Assam, Dibru river (4) India, West Bengal, Calcutta area (1)

Brahmaputra drainage; India, Assam Brahmaputra drainage; India, Assam Ganges, Brahmaputra, and Mahanadi drainages; Nepal, India, and Bangladesh Ganges, Brahmaputra, and Mahanadi drainages; Nepal, India, and Bangladesh Ganges, Brahmaputra, and Mahanadi drainages; Nepal, India, and Bangladesh Ganges, Brahmaputra, and Mahanadi drainages; Nepal, India, and Bangladesh Brahmaputra drainage; India, Assam Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Brahmaputra drainage; India, western Assam Mae Nam Khwae Noi drainage; Thailand Mae Nam Khwae Noi drainage; Thailand Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Upper Irrawaddy drainage; Myanmar Lower Irrawaddy, Sittang, middle Mekong; Thailand and coastal drainages in southern Myanmar, Mon State Lower Irrawaddy, Sittoung, middle Mekong; Thailand and coastal drainages in southern Myanmar, Mon State Southern Peninsular Thailand Southern Peninsular Thailand India, Assam India, Assam Brahmaputra drainage; India, Assam and northern West Bengal Upper Irrawaddy drainage, northern Myanmar Upper Irrawaddy drainage, northern Myanmar Upper Irrawaddy drainage, northern Myanmar Upper Irrawaddy drainage, northern Myanmar

Myanmar, Yangon Division, near Pyay (8) unknown Thailand, Phuket island (10) India, Assam India, Assam India, Assam (Aquarium trade) Myanmar, Myanmar, Myanmar, Myanmar,

Kachin Kachin Kachin Kachin

State, State, State, State,

Indawgyi lake (7) Myitkyina (6) Myitkyina (6) Indawgyi lake (7)

Indian subcontinent and Myanmar Peninsular Malaysia, Sundaland Mekong basin

L. Ru¨ber et al. / Molecular Phylogenetics and Evolution 32 (2004) 1010–1022

Genus

1012

Table 1 Summary of specimens, museum voucher numbers, ID = identification number, GenBank Accession numbers, sample locations, and distribution of the taxa used in this study

L. Ru¨ber et al. / Molecular Phylogenetics and Evolution 32 (2004) 1010–1022

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Fig. 2. Map of South-East Asia showing major changes in drainage basin morphology (adapted from Clark et al. (2004)). Map (A) shows the drainage pattern prior to the major captures, where the Upper Yangtze, Middle Yangtze, Upper Mekong, Upper Salween, and the Tsangpo rivers drained together to the South China Sea through the paleo-Red River (paleo-Red River drainage shown in bold black). (B) Capture/reversal of the Middle Yangtze into the Lower Yangtze (changes shown in bold black). (C) Capture of the Upper Yangtze river by the Middle Yangtze, and of the Upper Mekong and Upper Salween rivers into their modern drainage positions. The Tsangpo river was captured by the Irrawaddy river (changes shown in bold black). (D) Capture of the Tsangpo river through the Brahmaputra river into its modern course (changes shown in bold black). The boxed area refers to a more detailed map of this capture event (see Fig. 6) and the asterisk indicates the ‘‘Three River’’ area mentioned in the text. The indicated rivers are: B, Brahmaputra; I, Irrawaddy; M, Mekong; R, Red River; S, Salween; T, Tsangpo; and Y, Yangtze.

Only few studies have recognized the potential importance of changes in drainage basin morphology in understanding biogeographic patterns of the South East Asian Ichthyofauna (e.g., Kottelat, 1989). Furthermore, to our knowledge, thus far no phylogenetic studies have been conducted to test underlying vicariant speciation hypotheses. The former connection of the Tsangpo and the Irrawaddy rivers (Fig. 2C) may be important in understanding badid biogeography, and leads to a testable vicariant hypothesis. D. dario, as well as the B. badis and B. assamensis species groups are found in the Tsangpo–Brahmaputra–Ganges drainages, whereas the remaining two Dario species, B. kyar and the B. corycaeus species groups are found in the Upper Irrawaddy drainage (Fig. 1). Based on geological evidence for

large-scale changes in drainage systems (Fig. 2, Brookfield, 1998; Clark et al., 2004; Zeitler et al., 2001) we hypothesize an important vicariant event separating badid species found in the Tsangpo–Brahmaputra–Ganges drainage from species found in the Irrawaddy drainage. Unfortunately, neither molecular nor morphological phylogenies of badids exist that may allow to test this hypothesis. Thus, the aims of this study are (1) to establish a robust molecular phylogeny of the Badidae using both mitochondrial and nuclear DNA sequence data, and (2) to test a vicariant speciation hypothesis derived from geological evidence of a former Miocene connection of the Tsangpo river with the Irrawaddy drainage. This will allow us to evaluate the role of vicariance in shaping the current distribution patterns of badids.

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2. Methods

2.3. Phylogenetic analyses

2.1. DNA sources and extraction

Phylogenetic hypotheses of badid intra- and interspecific relationships (36 taxa) were based on complete mitochondrial cytb gene nucleotide sequences. We also performed separate interspecific (14 taxa) phylogenetic analyses of complete mitochondrial cytb, partial nuclear RAG1, and partial nuclear Tmo-4C4 gene nucleotide sequences. Finally, we performed a interspecific (14 taxa) phylogenetic analyses based on a combined cytb + RAG1 + Tmo-4C4 gene nucleotide sequence data set. We used the incongruence length difference (ILD) test (Farris et al., 1995) to test the null hypothesis of random partition between the different data sets using the partition homogeneity test in PAUP* v4.0b10 (Swofford, 2002) with 1000 replications. All uninformative characters were removed before the analyses. The hierarchical likelihood ratio test (LRT) implemented in MODELTEST v3.06 (Posada and Crandall, 1998) was used to determine the evolutionary model that best fit each of the data sets (Table 2). Inferred model parameters were used in maximum likelihood (ML) analyses, and to estimate ML distances for minimum evolution (ME) analyses. Maximum parsimony (MP) analyses were conducted with heuristic searches (TBR branch swapping, MULTREES option effective, and 10 random stepwise additions of taxa). Transversions (Tv) were given higher weights than transitions (Ts) based on empirical Ts:Tv ratios estimated from the ML trees (Table 2). Robustness of the inferred ME and MP trees was tested using non-parametric bootstrapping (Felsenstein, 1985) with 1000 pseudoreplicates. All the above mentioned phylogenetic analyses were conducted with PAUP* v4.0b10 (Swofford, 2002). Bayesian inferences (BI) of badid phylogeny were performed with MrBayes v3.03 (Huelsenbeck and Ronquist, 2001) based on the different data sets by launching Metropolis coupled Markov Chain Monte Carlo (MCMCMC) sampling for 1,000,000 generations (four simultaneous MC chains; sample frequency 100; burnin 50,000 generations; chain temperature 0.2) under the GTR + I + C model. For the combined cytb + RAG1 + Tmo-4C4 data set model parameters were estimated independently for the three data partitions using the ÔunlinkÕ command in MrBayes v3.03. We tested for base compositional biases using the v2 test as implemented in PAUP*. Alternative phylogenetic hypotheses were tested using the Shimodaira–Hasegawa test (SH; Shimodaira and Hasegawa, 1999) using RELL and 1000 bootstrap replicates. A LRT (Huelsenbeck and Crandall, 1997) was performed with ML trees with and without a molecular clock constraint for each data set. In addition, we tested constancy of evolutionary rates among taxa (two-cluster test) at the 5% significance level with RRTree v1.1 (Robinson-Rechavi and Huchon, 2000). In order to date major cladogenetic events a

To assess the molecular phylogeny of the Badidae, 33 individuals representing 12 out of 15 described species as well as one possibly undescribed species were utilized (Table 1). We were not able to obtain ethanol preserved material from B. chittagongis, B. ferrarisi (both belonging to the B. badis group) and D. dayingensis. In addition, three representatives of the genus Nandus (Teleostei, Nandidae), the sistergroup of the Badidae as supported by morphological (Kullander and Britz, 2002) and nucleotide sequence data (Ru¨ber et al., unpublished), were chosen as outgroups. Whole fish were preserved in 70–100% ethanol. Total genomic DNA was isolated from white muscle tissue or fin clips by proteinase K/SDS digestion, phenol–chloroform extraction, and ethanol precipitation (Kocher et al., 1989). 2.2. PCR amplification and sequencing The complete mitochondrial cytochrome b (cytb) gene was amplified with two versatile primers DonGlu F and DonThr R (Ru¨ber et al., 2004). In addition, for a subset of the taxa (interspecific data only), approximately 1500 bp of the nuclear RAG1 gene were amplified with primers R1-2533F and R1-4090R (Chen and Ortı´, submitted) and approximately 500 bp of the nuclear locus Tmo-4C4 were amplified with primers Tmo4C4F and Tmo-4C4R (Streelman and Karl, 1997). All PCR amplifications were conducted in 25 ll reactions containing 75 mM Tris–HCl (pH 9.0), 2 mM MgCl2, 0.4 mM of each dNTP, 0.4 lM of each primer, template DNA (10–100 ng), and Taq DNA polymerase (1 U, Biotools), using the following program: 1 cycle of 2 min at 94 C; 35 cycles of 60 s at 94 C, 60 s at 48– 54 C, and 90 s at 72 C; and finally, 1 cycle of 5 min at 72 C. After PCR purification using an ethanol/sodium acetate precipitation, samples were either cloned into pGEM-T vectors (Promega), and sequenced using M13 universal primers or were sequenced directly using PCR primers. Samples were cycle-sequenced with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (V3.0) in 10 ll reactions, and following manufacturerÕs instructions (Applied Biosystems), with 3.25 pmol of primer, 3 ll of Terminator Ready Reaction Mix, and 5% DMSO. The cycling profile for the sequencing reaction consisted of 25 cycles of 10 s at 96 C, 5 s at 50 C, and 4 min at 60 C. Cycle sequencing products were purified using MultiScreen plates (Millipore), and were analyzed on an ABI Prism 3700 DNA Analyzer (Applied Biosystems). Sequences have been deposited in GenBank under the Accession numbers given in Table 1.

L. Ru¨ber et al. / Molecular Phylogenetics and Evolution 32 (2004) 1010–1022

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Table 2 Summary of model and model parameters obtained by the hierarchical LRT implemented in MODELTEST, specification of data sets, details on the ML, MP, and ME analyses, and results of the hierarchical LRT test for constancy of rates of evolution and the relative rate test for the four different data sets used for the phylogenetic analyses Data set cytb 36 taxa Fig. 2

cytb 14 taxa Not shown

RAG1 14 taxa Fig. 3A

Alignment Invariant sites Informative sites

1140 563 533

1140 571 459

1473 1317 90

Model selected Prop. invar. sites a

TVM + I + C 0.45 1.33

TVM + I + C 0.45 1.37

HKY + C N/A 0.34

Empirical base frequency A C G T

0.29 0.33 0.11 0.27

0.28 0.32 0.12 0.28

0.27 0.23 0.27 0.23

0.30 0.18 0.26 0.26

Substitution rate Ts:Tv rate (MP Tv:Ts) A–C A–G A–T C–G C–T G–T

4.79a (5:1) 1.04 8.83 1.01 0.41 8.83 1.00

4.86a (5:1) 1.24 9.82 1.24 0.22 9.82 1.00

2.61 (3:1)

2.18 (2:1)

Base composition v2 Df P MP: Steps (number of trees) CI RI ME: score ML: ln ML (clock): ln 2D Relative rate test (Nandus (Dario, Badis)) DK P a b c *

75.13 87 0.81

22.33 39 0.98

4273 (1) 0.51 0.81 3.56 9438.13 9459.11 41.96*,b

3566 (1) 0.58 0.58 3.20 7747.18 7757.99 21.62*,c

0.056* 0.00

0.065* 0.00

– – – – – –

Tmo-4C4 14 taxa Fig. 3B 511 426 40 HKY + C N/A 0.27

– – – – – –

2.14 39 1.00 271 (2) 0.94 0.92 0.13 3103.47 3109.03 11.12c

0.012* 0.04

5.16 39 1.00 138 (3) 0.93 0.91 0.25 1291.69 1298.07 12.76c

0.008 0.54

Combined 14 taxa Fig. 3C 3124 2314 589 GTR + I + C 0.52 0.43

0.27 0.25 0.22 0.26

3.71a (4:1) 2.75 8.15 2.57 0.54 19.81 1.00

9.46 39 0.99 3619 (1) 0.62 0.61 1.13 12787.73 12797.43 19.40c

0.065* 0.00

Estimated from ML tree. (v2(28,0.05) = 41.33). (v2(12,0.05) = 21.02). p < 0.05.

linearized tree based upon the cytb data set (ML tree) was constructed using the non-parametric rate smoothing (NPRS) method (Sanderson, 1997) as implemented in TreeEdit v1.0 (Rambaut and Charleston, 2001). In order to estimate the magnitude of error from substitutional noise, confidence intervals were obtained by parametric bootstraping. Seq-Gen v1.2.2 (Rambaut and Grassly, 1997) was used to simulate 500 parametric bootstrap data sets (1140 bp long) along the original ML

tree under the TVM + I + C model (Table 2). For each parametric bootstrap data set the ML tree was reconstructed in PAUP* v4.0b10 and converted into an linearized tree in TreeEdit v1.0 as described above. Mean and 95% confidence interval for each node depth was then calculated. To roughly estimate divergence times between major clades and in the absence of a fossil record, we calibrated the age of the most recent common ancestor of the B. badis species group using a cytb substitution

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L. Ru¨ber et al. / Molecular Phylogenetics and Evolution 32 (2004) 1010–1022

rate of 8.2 · 10 9 (substitutions · base pair 1 · year 1), which corresponds to a pairwise sequence divergence of 1.64%/Myr. This substitution rate was derived for the same gene for European cyprinids based upon two independent, and well-dated geological events (Zardoya and Doadrio, 1999).

3. Results 3.1. Phylogenetic analyses Specifications of the different data sets used for the phylogenetic analyses, evolutionary models applied, as well as ML (non-clock, clock), MP, and ME scores are given in Table 2. No base compositional biases were observed (Table 2). The partition homogeneity test detected significant congruence between all data partitions (cytb vs. RAG1, P = 0.71; cytb vs. Tmo-4C4, P = 0.64; RAG1 vs. Tmo-4C4, P = 1.0; and mtDNA vs. nucDNA, P = 0.12). The alignment of cytb gene nucleotide sequences of 33 Badidae specimens revealed 27 unique haplotypes (Table 1). The Bayesian phylogenetic tree based on the cytb nucleotide sequences is shown in Fig. 3. MP phylogenetic analyses with a 5:1 Tv:Ts weighting scheme resulted in one shortest tree (Table 2) with the same branching pattern. Different Tv:Ts weighting schemes (e.g., 1:1, 2:1) arrived at identical MP tree topologies (not shown). ME and ML trees showed identical topologies to the BI and MP trees (Fig. 3). Within the Badidae, two main lineages corresponding to the genera Dario and Badis, respectively, were recovered (Fig. 3). In order to facilitate the discussion of our results we follow the species group assignment of Kullander and Britz (2002). Within the genus Badis five clades, corresponding to the five Badis species groups defined by Kullander and Britz (2002), were recovered (Fig. 3). The B. corycaeus species group was identified as sistergroup to the B. badis and B. assamensis species groups. B. kyar was consistently placed as sister group to the B. ruber species group with high posterior probability and bootstrap support values (Fig. 3). We also performed four separate phylogenetic analyses (based on cytb, RAG1, Tmo-4C4, and a combined gene nucleotide sequence data set) for a restricted taxon-sampling set including only interspecific data (13 Badis and Dario species plus one Nandus as outgroup). The range of uncorrected sequence divergence for these data sets were: within Badis (8.16–22.02%/0.39–1.36%/0.78– 1.76%; for the cytb, RAG1, and Tmo-4C4, respectively) and between Badis and Dario (25.44–29.56%/5.16– 6.38%/8.22–9.39%). The phylogenetic analyses using the 14-taxon cytb data set (Table 2) rendered congruent results (trees not shown) reflecting identical interrelationships to those obtained using the 36-taxon cytb data set.

The phylogenetic analyses based on the nuclear RAG1 nucleotide sequences (Table 2) using the Bayesian inference method resulted in the phylogenetic tree shown in Fig. 4A. BI as well as MP, ME, and ML phylogenetic analyses supported a clade consisting of B. kyar, and the B. badis and B. assamensis species groups (the relative phylogenetic position of B. kyar within this clade was unresolved in all analyses due to low statistical support). The phylogenetic analyses based on the nuclear Tmo4C4 nucleotide sequences (Table 2) using the Bayesian inference method resulted in the phylogeny shown in Fig. 4B. MP, ME, and ML yielded identical topologies. In these phylogenetic analyses, the clade consisting of B. kyar, and the B. badis and B. assamensis species groups was also recovered with moderately high statistical support. The well-supported sistergroup relationship of B. corycaeus and B. pyema found in all previous phylogenetic analyses was not recovered with Tmo-4C4. In contrast to the results obtained with the cytb and RAG1 data sets, B. khwae was resolved as the most basal lineage within the B. ruber species group in all analyses using Tmo-4C4, although with low bootstrap support in the MP and ME analyses (Fig. 4B). All phylogenetic analyses based on the combined mitochondrial and nuclear nucleotide sequences (Table 2) resulted in identical topologies (Fig. 4C) to those recovered in the cytb analyses. In all cases, B. kyar was resolved as sister group to the B. ruber species group. The statistical support of the recovered phylogenetic hypothesis was stronger in the analyses based on the combined data set than in those based on each data partition separately. We used the SH tests to evaluate alternative phylogenetic hypotheses that resulted from different placements of B. kyar within Badis and of different relationships within the B. ruber group depending on the data set used (Table 3). All alternative topologies to the cytb and the combined data set were clearly rejected by these two data sets. The RAG1 data set only rejected one alternative topology in which B. kyar was placed as sistergroup to the B. ruber group. With the Tmo-4C4 data set, no alternative hypotheses could be rejected (Table 3). 3.2. Rates of evolution and divergence time estimates Likelihood ratio tests with and without the molecular clock enforced rejected overall constancy of rates of evolution in the Badidae for the two cytb data sets, but not for the RAG1, Tmo-4C4, and combined data set (Table 2). All data sets showed longer branches for Dario than Badis (only shown for cytb; Fig. 5A). To further explore rate heterogeneity we used the relative rate test and found significant differences in rates between Dario and Badis for cytb, RAG1, and the combined data sets (Table 2). In the absence of constant rates of evolution

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Fig. 3. Reconstructed phylogeny of the Badidae using a Bayesian phylogenetic approach. Three Nandidae taxa were utilized as outgroups. The number above each branch refers to the Bayesian posterior probability (shown as percentage) of the node derived from 9500 MCMCMC sampled trees on the basis of complete cytb mitochondrial DNA nucleotide sequence data (1140 bp). Bootstrap values (>50%) for MP and ME (upper and lower values, respectively) are shown below branches. Major Badis clades are highlighted with black boxes and correspond to the species group designation of Kullander and Britz (2002). Code numbers behind species names correspond to ID numbers given in Table 1. The numbers and names in brackets refer to the localities listed in Table 1; (a) see Table 1 for more details.

for the cytb gene we reconstructed a linearized tree using NPRS based on the 14-OTU data set (Fig. 5B). To transform relative times into absolute ages the NPRS tree was calibrated using a teleost cytb substitution rate of of 8.2 · 10 9 (substitutions · base pair 1 · year 1; see Section 2). Tentative divergence dates for the main cladogenetic events within the Badidae were: an Eocene origin of the family, a Mid Oligocene origin of the genera Badis and Dario; and a majority of speciation events within the genus Badis in the Miocene (Fig. 5B). We used parametric bootstrapping to calculate the 95% confidence intervals in order to account for the stochastic behavior of substitutional processes. The branch length of the original tree (ML) used for the parametric bootstrapping was always contained within the 95% confi-

dence intervals. Yet, we noted that the 95% confidence intervals for some basal nodes were biased towards the present compared to the original ML tree (Fig. 5B). Within the badids a putative vicariant event that involves the separation between Brahmaputra–Ganges– Mahanadi and the Upper Irrawaddy-clades can be inferred (Fig. 5B). This vicariant event may be responsible for the separation between D. dario and D. hysginon, as well as between the B. badis + B. assamensis and the B. corycaeus species groups (Fig. 5B). The coinciding 95% confidence intervals in the Oligocene–Miocene boundary (19–24 Mya) indicate that the same vicariance event might have been responsible for the separation of the Brahmaputra and Upper Irrawaddy lineages of Badis and Dario, respectively.

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Fig. 4. Phylogenetic reconstruction of the Badidae with different nucleotide sequence data sets using a Bayesian phylogenetic approach. For all analyses the nandid Nandus nandus was used as outgroup. Phylogenetic hypotheses were derived from (A) the nuclear gene RAG1 (1476 base pairs), (B) the nuclear loci Tmo-4C4 (511 base pairs), and (C) from a combined analysis of a total of 3124 base pairs of mitochondrial (cytb) and nuclear (RAG1 and Tmo-4C4) nucleotide sequence data. The number above each branch refers to the Bayesian posterior probability (shown as percentage) of the node derived from 9500 MCMCMC sampled trees. Bootstrap values (>50%) for MP and ME (upper and lower values, respectively) are shown below branches. The phylogenetic position of Badis kyar is highlighted in bold.

Table 3 Testing of alternative phylogenetic hypotheses within the Badidae using the Shimodaira–Hasegawa test Topology

cytb

RAG1

Tmo4-C4

Combined

ky(si,kh,ru) ky(ka,ba,sp) ky(ka,ba,sp,as,bl) si(kh,ru) kh(si,ru)

7747.178 7807.586*** 7797.528** 7747.178 7764.515*

3141.812* 3103.471 3104.384 3103.471 3103.471

1299.175 1294.674 1291.690 1295.709 1291.690

12787.726 12857.366*** 12837.814*** 12787.726 12803.658*

ky, B. kyar; ka, B. kanabos; ba, B. badis; sp, B. sp. ‘‘Assam’’; as, B. assamensis; bl, B. blosyrus; si, B. siamensis; kh, B. khwae; and ru, B. ruber. The ML hypothesis is underlined. * p < 0.05. ** p < 0.01. *** p < 0.001.

4. Discussion 4.1. Intrarelationships of the Badidae Phylogenetic studies based on multiple loci reveal a more complete picture of the evolutionary history of a group of closely related species than those solely based on a single locus (e.g., Machado and Hey, 2003; Rokas et al., 2003). Here, we studied the phylogenetic history of the family Badidae based upon one mitochondrial (cytb) and two nuclear (RAG1 and Tmo-4C4) genes. Our phylogenetic analyses provided a robust framework for badid intrarelationships. All genes validated with high statistical support the split of the family into two genera Badis and Dario. Moreover, the division of the genus Badis into several species groups as proposed by Kullander and Britz (2002) was also well supported. The only disagreement between phylogenies derived from different loci was the placement of B. kyar. In

the cytb-based phylogeny the position of B. kyar, a species that could not be assigned to any species group by Kullander and Britz (2002), was found to be the sister group of the B. ruber species group (Fig. 1). In contrast, in the nuclear-based phylogenetic hypotheses, B. kyar was placed consistently in a clade that included the B. badis and B. assamensis species groups (Fig. 4). No alternative phylogenetic hypotheses tested could be rejected with the Tmo-4C4 data set, whereas both the cytb and the RAG1 data sets each rejected reciprocally alternative placements of B. kyar (Table 3). Species level phylogenies are often difficult to reconstruct due to low levels of genetic divergence, and hence a lack of sufficient synapomorphies for establishing reliable sister group relationships. The two nuclear genes RAG1 and Tmo-4C4 showed very few informative sites to resolve the Badidae phylogeny (Table 2). When only considering the genus Badis the RAG1 and the Tmo4C4 genes revealed 20 and merely nine informative sites,

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Fig. 5. Rates of evolution and molecular clock. (A) ML phylogram using complete mitochondrial cytb nucleotide sequences (outgroup not shown). Note the relatively longer branches of the genus Dario with respect to the genus Badis. The numbers and names in brackets refer to the localities listed in Table 1; (a) see Table 1 for more details. (B) Ultrametric ML tree based on the NPRS transformation using the same data set as above. Black bars represent the 95% confidence intervals derived from 500 parametric bootstrap data sets. The scale bar below the tree shows the time scale resulting from a calibration of the cytb molecular clock using a substitution rate of 8.2 · 10 9 (substitutions · base pair 1 · year 1) based on the node highlighted with a star that showed a mean TVM + I + C distance of 12% (= 7.3 Myr). The substitution rate was derived from the complete cytb of European cyprinids based upon the data given in Zardoya and Doadrio (1999; range 7.4 · 10 9–9.2 · 10 9; mean 8.2 · 10 9). Geographic location of species is indicated by symbols before species names: (back squares) Brahmaputra, Ganges, and Mahanadi; (grey circles) Upper Irrawaddy; and (unfilled triangles) Lower Irrawaddy and Peninsular Thailand (see Fig. 1). The overlap of the 95% confidence intervals for the suggested vicariant events (19–23 Myr ago) separating the Brahmaputra and the Upper Irrawaddy clades is indicated with a grey box.

respectively (not shown). Hence, we feel that at present it is premature to draw any firm conclusion about the conflicting phylogenetic placement of B. kyar resulting from our comparative analyses of mitochondrial and nuclear genes. Although either retention of ancient polymorphisms or introgressive hybridization might have caused the observed pattern, the low level of sequence divergence observed in the two nuclear genes render these scenarios speculative. Simulation studies have shown, that the accuracy of tree reconstruction declines fast with low substitution rates (Yang, 1998) as those observed in the nuclear RAG1 and Tmo-4C4 data sets (Table 2). Undoubtedly, more nuclear gene sequence data will help to resolve the conflicting phylogenetic position of B. kyar within badids. 4.2. Differences in rates of molecular evolution For all gene fragments analyzed the genus Dario showed longer branches than Badis (see e.g., Fig. 5A). The relative rate test revealed a significant increase in rates of substitutions in Dario in all data sets except Tmo-4C4 (Table 2). There are generally three hypotheses invoked to account for rate increase in an evolutionary lineage (1) lower DNA repair efficiency, (2) higher metabolic rate, and (3) faster generation time (Martin

and Palumbi, 1993). Dario species are sexually mature at a much smaller size than Badis species (standard length 15.2–21.2 mm vs. 28.5–67.6 mm). Therefore, differences in generation time are particularly persuasive in explaining the observed differences in substitution rate between the two genera. 4.3. Biogeography and divergence time estimates Our study is the first published use of phylogenetic data, to our knowledge, to test vicariant speciation hypotheses derived from geological evidence of large-scale changes affecting the Irrawaddy and Tsangpo–Brahmaputra drainages during the Miocene. Using a cytb substitution rate of 8.2 · 10 9 (substitutions · base pair 1 · year 1), we calibrated our molecular phylogenetic hypothesis of the Badidae (Fig. 5). According to the estimated dates, badids likely originated in the Eocene (about 47 Mya). Several vicariant events are apparent in the Badidae (Fig. 5). We observed geographic splits within two clades of species inhabiting the Brahmaputra–Ganges drainages and the upper Irrawaddy drainages. This pattern was observed in D. dario vs. D. hysginon and between B. pyema + B. corycaeus vs. B. kanabos + B. badis + B. sp. ‘‘Assam’’ + B. assamensis + B. blosyrus (Fig. 5). After NPRS linearization, we noted

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that the origin of the former clade predates that of the latter by about 10 Myr, but this may be an artifact of the analyses due to the extremely fast rate of evolution of Dario species. In fact, our analyses show that the 95% confidence intervals of these two cladogenetic events overlapped in a timeframe in the Late Oligocene–Early Miocene (19–23 Mya, Fig. 5). Since Gregory and GregoryÕs (1923) pioneering work, changes in drainage basin morphology have been considered important factors influencing biogeographic patterns of the South East Asian Ichthyofauna (e.g., Kottelat, 1989). A recent compilation of the drainage history in southeastern Tibet identified river captures and drainage direction reversals, prior to or coeval with the initiation of Miocene uplifts in Eastern Tibet, as the main factors affecting paleodrainages (Clark et al., 2004). While Gregory (1925) proposed a paleo connection of the Tsangpo to the Chindwin and Lower Irrawaddy more recent data indicate a paleo Tsangpo–Upper Irrawaddy connection (Brookfield, 1998; Clark et al., 2004; Zeitler et al., 2001, Fig. 6A). According to Clark et al. (2004) the paleo-Tsangpo was connected through the Parlung river (which then flowed southeast) to the Upper (and Lower) Irrawaddy river (Fig. 6B). The paleo-Tsangpo–Parlung river was then captured by the Upper Lhuit river (a tributary of the Brahmaputra river), and separated from the Irrawaddy. More recently, the paleo-Tsangpo–Parlung river was disconnected from the Upper Lhuit river, and captured by the Upper Brahmaputra river (a short, steep transverse Himalayan river at that time) resulting in the current drainage systems (Fig. 6C). Our hypothesis of a vicariant event between the formerly connected Tsangpo and Upper Irrawaddy (Fig. 6), which is mirrored twice within the Badidae (in Badis and Dario) is further supported by data from anabantoid species pairs occurring in North East India vs. Myanmar (Ru¨ber et al., unpublished). The uncorrected cytb sequence divergence between the B. badis and B. assamensis species group vs. the B. corycaeus species group is 18.98 ± 0.49 (range 18.24–19.56). Similar levels of cytb divergence were found among anabantoid species that show comparable allopatric distributions (e.g., Colisa labiosa (Myanmar) vs. the remaining Colisa spp. (India) 17.15–18.04; and Ctenops (India) vs. Parasphaerichthys (Myanmar) 20.17–21.6; Ru¨ber et al., unpublished). Although preliminary, these data indicate that the levels of cytb sequence divergence between species pairs occurring in the Brahmaputra–Ganges drainages and the Irrawaddy drainage are in the same range. Badid clades from the Brahmaputra–Ganges drainages are effectively separated from Irrawaddy clades by the Indoburman ranges, a Miocene orogen related to the collision of the Indian plate with the West Burma Block (Bannert and Helmcke, 1981; Bender, 1983; but see Nandy (1976) who gives an Eocene age for the initial

Fig. 6. Maps showing the current drainage systems in the IndoBurman region and reconstruction of the paleo connection of the Tsangpo with the Irrawaddy (adapted from Clark et al., 2004). (A) overview map of North Myanmar, Bangladesh, and North East India showing the location of the Indoburman ranges (striped line in grey). Symbols indicate the three main geographic distributions of clades recovered in the phylogenetic analyses (see also Fig. 4): (black square) Brahmaputra, Ganges, and Mahanadi; (grey circle) Upper Irrawaddy; and (unfilled triangle) Lower Irradaddy and Peninsular Thailand. The black box shows the location of the maps shown in (B) and (C). Map (B) shows the reconstruction of the Tsangpo– Brahmaputra river capture. Prior to capture the paleo-Tsangpo river flowed into the Irrawaddy river. The Tsangpo river was then likely fist captured by the Lhuit river then subsequently by the Brahmaputra river (Clark et al., 2004). The two capture points are indicated by grey bars and arrows represent flow directions. Map (C) shows the present drainage systems. The doted line in grey denotes the separation between the headwaters of the Irrawaddy–Chindwin and the Brahmaputra drainages. The indicated rivers are: B, Brahmaputra; C, Chindwin; G, Ganges; I, Irrawaddy; L, Lhuit; M, Mahanadi; P, Parlung; and T, Tsangpo.

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uplift of the South Western Indoburman range). Progressive uplifting of the Indoburman ranges started in the South West (Arakan-Yoma mountains) towards North East (Naga Hills, Patakai Ranges), thus separating the Inner-Burman Tertiary Basin (Chindwin Basin) from the Bay of Bengal (Bannert and Helmcke, 1981; Bender, 1983; Verma and Krishna Kumar, 1987). Although merely speculative, it seems possible that the Miocene uplift of the Indoburman ranges might also have played an important role in shaping the current distribution patterns of badids. Besides the great affinities of the fish faunas between the Brahmaputra, Tsangpo, Chindwin, and Irrawaddy (Kottelat, 1989) and the hypothesized paleo connection between the Tsangpo–Brahmaputra and Irrawaddy drainages (Brookfield, 1998; Clark et al., 2004; Gregory, 1925; Zeitler et al., 2001, Fig. 6) there is little data available that would allow to reconstruct the paleo-freshwater river systems in Myanmar and adjacent regions. Paleogeographical maps (Brunnschweiler, 1974) indicate that the area of Assam and the Inner-Burman Tertiary Basin (Chindwin Basin) have progressively developed into a shallow marine sedimentary basin since the late Cretaceous with its largest extension in the Mid Eocene to Early Oligocene and suggest that the actual major freshwater river systems in North East India and Myanmar probably only exist since the Miocene. However, more paleobiogeographic reconstructions are needed to better understand the potential role of Miocene tectonic events shaping paleodrainages on the current distribution pattern of freshwater fishes in North East India–Myanmar. In conclusion, our data provide the first molecular phylogenetic (mtDNA and nucDNA) framework of Badidae intrarelationships, and further support the division of badids into species groups as suggested by morphological evidence (Kullander and Britz, 2002). The phylogenetic position of B. kyar was conflicting between different data partitions, and thus awaits the incorporation of more nuclear sequence data. Our molecular evidence tentatively highlights the importance of paleo river connections between the Brahmaputra and the Irrawaddy drainages (Clark et al., 2004; Gregory, 1925) for understanding the evolution of the Badidae. According to our results, early cladogenesis both within the genera Dario and Badis were likely prompted by a vicariant event that separated Brahmaputra and Irrawaddy drainages. Calibration of a molecular clock allowed us to date the proposed vicariant scenario at the Oligocene–Miocene boundary (19–24 Mya), which seems to agree with geological evidence for the separation of these drainages caused by tectonic uplifts in Eastern Tibet. These geological events appear to have played a major role in the diversification of badids thus supporting the hypothesis of primarily vicariant-based speciation in this group. However, given the current uncertainty in the paleobiogeographic reconstruction

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of North East India and Myanmar, establishing temporal congruence between potential vicariant and cladogenetic events remains provisional. We expect new paleobiogeographic insights from broader comparative phylogenetic studies of freshwater fishes, from the Indo-Burma region (North East India and Myanmar), one of the worldÕs leading biodiversity hotspots (Myers et al., 2000). Acknowledgments We are grateful to M. Kottelat (Cornol, Switzerland) and F. Scha¨fer (Rotgau, Germany) for providing some of the samples. Specimens were collected during field surveys supported by grants to F. Fang from the Hierta-Retzius Foundation (the Royal Swedish Academy of Sciences), the Ax:son Johnson Foundation, and the Riksmusei Va¨nner, and to SOK from the Swedish Natural Science Research Council (R-RA 04568-316). We are indebted to K. Lahkar (Dibrugarh University), U Win Aung, and U Tun Shwe (Fisheries Department, Union of Myanmar), U Mya Thein Roy (Myanmar Aquaculture and Fisheries Association), T. Win, and A. Roos (NRM) for field assistance and permits. We also thank W.J. Chen and G. Ortı´ for permission to use the RAG1 primer sequences prior to publication. We thank M.K. Clark, J.R. Curray, L.H. Royden, and A. Uddin for providing invaluable help regarding the geological history of North East India and Myanmar. This study was completed while R.B. held a Ôsenior visiting scientistÕ fellowship of the Office of Fellowships and Grants, Smithsonian Institution, National Museum of Natural History, Division of Fishes, Washington D.C. This research was supported by a BIOD-IBERIA grant at the Museo Nacional de Ciencias Naturales under the European Commission HUMAN POTENTIAL PROGRAM to R.B. and S.O.K. L.R. was supported by the Swiss National Science Foundation postdoctoral fellowship 823A-061218. A grant from the Janggen-Po¨hn-Foundation, Switzerland to L.R. funded laboratory work. References Bannert, D., Helmcke, D., 1981. The evolution of the Asian plate in Burma. Geologische Rundschau 70, 446–458. Barlow, G.W., Liem, K.F., Wickler, W., 1968. Badidae, a new fish family—behavioural, osteological, and developmental evidence. J. Zool. Lond. 156, 415–447. Bender, F., 1983. Geology of Burma. Borntra¨ger, Berlin. Brookfield, M.E., 1998. The evolution of the great river systems of southern Asia during the Cenozoic India–Asia collision: rivers draining southwards. Geomorphology 22, 285–312. Brunnschweiler, R.O., 1974. Indoburman Ranges. In: Spencer, A.M. (Ed.), Mesozoic-Cenozoic Orogenic Belts. Geol. Soc. London, Spec. Publ., London, pp. 279–299. Clark, M.K., Schoenbohm, L.M., Royden, L.H., Whipple, K.X., Burchfiel, B.C., Zhang, X., Tang, W., Wang, E., Chen, L., 2004.

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