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An alternative source of time constraints in teleostean phylogeny by evaluating a …

Biology Articles » Biogeography » Mitogenomic evaluation of the historical biogeography of cichlids toward reliable dating of teleostean divergences » Results and discussion

Results and discussion
- Mitogenomic evaluation of the historical biogeography of cichlids toward reliable dating of teleostean divergences

Mitochondrial genomes of cichlids

We determined complete or nearly complete mtDNA nucleotide sequences for six new cichlids from Africa, South America, Madagascar, and Indo/Sri Lanka (Table 1). The sizes of these genomes ranged from 16,457 to 16,556 bp, including approximately 800 bp in the control region. Tylochromis polylepis alone appears to have a somewhat longer control region (approximately 1200 bp) although the exact sequence of the region was unable to be determined because of the long poly-T sequences within the region. We also analyzed the previously published mitogenomic sequences of four cichlid species (Table 1). Oreochromis mossambicus (accession no. AY597335) was not included because a congeneric taxon (Oreochromis sp.) sequenced by Mabuchi et al. [43] had already been sampled.

All 37 genes encoding two rRNAs, 22 tRNAs, and 13 proteins were identified in these 10 cichlid mitogenomes, basically in the same order and orientation found for most other vertebrates. Transfer RNA genes could be folded into secondary structures typical of vertebrate mitochondrial tRNA [29]. The base composition of cichlid mitogenomes was skewed (data not shown) similarly to those of other vertebrates [44].

Phylogenetic relationships

Figure 1 shows the phylogenetic relationships inferred from the Bayesian analysis among the 52 bony fishes, estimated with two sharks as an outgroup. The tree topology was identical to that obtained by the partitioned ML analysis (data not shown). These bony fish taxa included two sarcopterygians (coelacanth and lungfish), nine basal actinopterygians (polypterids, acipenseriforms, lepisosteids, and amiid), and 41 teleosts, including 10 cichlids. The phylogenetic relationships obtained for non-cichlid taxa (Fig. 1) were largely consistent with those from previous mitogenomic studies [28,43,45], except for a difference in the sister group of holosteans (lepisosteids and amiid).

Although Inoue et al. [28] suggested that the "Ancient Fish Clade" unites acipenserids, lepisosteids, and amiid, our phylogenetic analysis supports the neopterygian clade (lepisosteids + amiid + teleosts), in agreement with an analysis of nuclear DNA sequences [46] and morphological characters [47]. Relationships between the basal actinopterygians and teleosts were not stable against changes in taxonomic representations and the genes used and varied between the two hypotheses (data not shown). We tentatively assumed the neopterygian relationship for subsequent analyses because this was consistent in both morphological and molecular (based on mitochondrial and nuclear sequences) analyses. However, we also conducted analyses to evaluate how our major conclusions in dating depend on the two alternative phylogenetic relationships (Table 3).

Table 3. Comparison of divergence time estimates between different time constraints and studies

In terms of the relationships among 20 percomorphs containing 14 labroids (two labrids, two pomacentrids, and 10 cichlids), we reconfirmed the polyphyly of Labroidei [43] whereby labrids (designated Labroidei 1 in Fig. 1) and cichlids + pomacentirids (Labroidei 2) appear in separate lineages of teleosts. The non-monophyly of the labroid taxa was supported by a number of nodes with 100% posterior probability and 100% bootstrap values (Fig. 1).

Figure 1. A Bayesian tree based on mitogenomic DNA sequences. This is a 50% majority rule consensus tree among 10,000 pooled trees from two independent Bayesian MCMC runs. The data set comprises aligned gap-free nucleotide sequences of 10,034-bp length from 54 taxa, which included 4,887 variable sites and 3,936 parsimony-informative sites. Partitioned Bayesian analyses were conducted using the GTR + I + Γ model and with all model parameters variable and unlinked across partitions. The numerals at internal nodes or branches indicate Bayesian posterior probabilities (left) and maximum likelihood bootstrap probability values (right) from 1000 replicates, respectively (shown as percentage for values above 50%).

Among the 10 cichlid taxa that we used, four were from Africa, two from South America, three from Madagascar, and one from Indo/Sri Lanka. The tree (Fig. 1) supports the monophyly of Cichlidae and two other continental groups from Africa and South America. Four basal taxa from Madagascar and Indo/Sri Lanka are not monophyletic, and two (Paretroplus from Madagascar and Etroplus from Indo/Sri Lanka) corresponding to Etroplinae sensu Sparks and Smith [16] form a sister group to all other cichlids. The other two Malagasy taxa (Paratilapia and Ptychochromoides), corresponding to Ptychochrominae sensu Sparks and Smith [16], form a sister group to the African + Neotropical clade. These results are consistent with previous molecular studies that used a few mitochondrial or nuclear gene sequences [14-16,48], as well as morphological studies [13].

However, these previous studies did not fully evaluate the statistical significance in rejecting alternative hypotheses of cichlid relationships. We conducted KH and SH tests, as well as a test using Bayes factor. Based on these tests, alternative hypotheses assuming the monophyly of Malagasy + Indo/Sri Lankan cichlids (constraint 1), Old World cichlids (constraint 2), and African + Indo/Sri Lankan cichlids (constraint 3) are all very unlikely (Table 4). These results provide statistical support for the paraphyletic assemblage of the Malagasy + Indo/Sri Lankan taxa to the African + Neotropical clade.

Table 4. Test of alternative phylogenetic hypotheses for continental cichlid groups

If Cichlidae originated in Cenozoic Africa and migrated into South America, Madagascar, and India via saltwater dispersal [19,49], Malagasy/Indo Sri Lankan and/or Neotropical taxa would probably be nested in the African clade, and alternative relationships (e.g., those corresponding to constraints 2 and 3) would likely appear. However, these relationships were not found, thus supporting the vicariant divergence scenario [13,14,18], at least from a topological standpoint.

Timing of cichlid divergences

We conducted divergence time estimation among 54 bony fishes, including 10 cichlids (Fig. 2). Twenty-one time constraints based on extensive fossil evidence for bony fishes (Table 2) were used. Following the advice of Benton and Donoghue [2] to set fossil-based time constraints as hard lower boundaries and soft upper boundaries, we chose older values for upper boundaries. We estimated the divergence between African + Neotropical cichlids and Malagasy + Indo/Sri Lankan (ptychochrominae) cichlids to be approximately 96 MYA (78–115 MYA at 95% credibility). The divergences of African vs. Neotropical cichlids and Malagasy vs. Indo/Sri Lankan cichlids within the Etroplinae were estimated to be approximately 89 MYA (72–108 MYA) and 87 MYA (69–106 MYA), respectively.

Figure 2. Divergence times estimated from the partitioned Bayesian analysis. A posterior distribution of divergence times with 95% credibility intervals (shaded rectangles) was obtained using mitogenomic DNA sequences (10,034 sites). Two sharks (Scyliorhinus canicula and Mustelus manazo) were used as an outgroup (not shown). The multidistribute program [41] was used to estimate divergence times assuming the tree topology shown in Fig. 1. Letters indicate nodes at which maximum and/or minimum time constraints were set (see Table 2 for details of the individual constraints). Paleogeographical maps at 148 MYA, 120 MYA, 95 MYA, and 85 MYA [50] are shown. Dark-gray areas on the maps represent those being fragmented within Gondwanaland at those times.

We then compared the estimated divergence times among cichlids and the probable times of continental fragmentation based on geological evidence. The divergence time between Malagasy and Indo/Sri Lankan taxa within Etroplinae (~87 MYA: 69–106 MYA) is very close to the time of separation between Madagascar and India (85–95 MYA) [50,51]. The divergence time estimated between African and Neotropical clades (~89 MYA: 72–108 MYA) is also close to the time of separation between African and South American landmasses (~100 MYA) [50,51]. The divergence time between African + Neotropical cichlids and Malagasy ptychochrominae cichlids (~96 MYA: 78–115 MYA) appears to be somewhat more recent than the time generally accepted for the complete separation of the Indo-Madagascar landmass from Gondwanaland (120–130 MYA) [50,51]. However, some studies [52] have postulated an extended connection between India and Antarctica by approximately 112 MYA, which is within the 95% credibility range for the African/Neotropical vs. ptychochrominae cichlid divergence. Taken together, these results are consistent with the vicariant divergence of continental cichlid groups during Cretaceous times and argue against their Cenozoic dispersal.

Vences et al. [19] calibrated a molecular clock for cichlids that assumed that the divergence time of the most basal endemic lineages in East African Rift lakes (e.g., Tanganyika) corresponds to the geological estimate of the age of the lakes. These estimated divergence times between continental cichlid clades were all in the Cenozoic (rather than the Mesozoic, as we demonstrate in Fig. 2) and supported the hypothesis of long-distance Cenozoic transmarine dispersal of cichlids. This view of the Cenozoic (or latest Cretaceous) origin and transmarine dispersal of cichlids has also been supported by some biogeographers [49] because it is consistent with cichlid fossil records, which first occur in South America and Africa in the Eocene [20,53]. However, the clock-based dating procedures of Vences et al. [19] present some problems. The strict molecular clock may not hold for all cichlid lineages [15], and the premise that the oldest endemic cichlid divergence is synchronized with the formation of the lakes may not be valid. Some lineages that had diverged outside the lake may have immigrated in parallel [7]. In addition, there is no definitive, geologically based time estimate for the formation of the lakes.

More recently, Genner et al. [7] used two mitochondrial (cytochrome b and 16S rRNA) and one nuclear (TMO-4C4) gene fragments to estimate the divergence times among cichlids. When the cichlid divergence by Gondwanan vicariance was assumed, the resultant divergence times were more consistent with those estimated with time constraints from previous paleontological and molecular studies [2,54-57] than when the Cenozoic cichlid divergence was assumed based on fossil records.

Although we concur on the Gondwanan origin and vicariant divergence of cichlids, Genner et al. [7] evaluated this biogeographic hypothesis somewhat indirectly, in that the fitness of estimated times of cichlid divergences to those obtained with time constraints from previous studies was qualitatively compared between alternative assumptions on cichlid biogeography. We evaluated cichlid divergence times more directly by using longer mitogenomic sequence data and dozens of non-cichlid taxa, allowing us to set many time constraints purely from the paleontological data and providing additional evidence for an ancient cichlid divergence on Gondwanaland, despite the general paucity of the Mesozoic and Cenozoic paleontological record on bony fishes.

Gondwana fragmentation as time constraints

In Figure 3, minimum time constraints based on fossil records (see Table 2) are plotted against molecular time estimates of the corresponding divergences (values taken from Fig. 2). In this figure, minimum age estimates of Gondwanan fragmentations are also plotted against the corresponding molecular time estimates of continental cichlid groups. It should be noted here that the latter data points reflecting Gondwanan fragmentation history (closed triangles) are plotted well on the line of 1:1 relationship whereas most of the data points reflecting fossil records (closed circles) are considerably below the line of the 1:1 relationship. This pattern suggests that Gondwana fragmentation history that is congruent with the cichlid phylogeny can be effective time constraints better than most of the Mesozoic and Cenozoic fossil records used here.

Figure 3. Comparison of paleontological and molecular estimates of divergence times. Minimum estimates of divergence times deducible from fossil records (see Table 2) were plotted as closed circles against molecularly estimated divergence times (mean values for the divergence times shown in Fig. 2). Closed triangles show plots of the timing of continental breakups against the molecular time estimates of cichlid divergences between the corresponding continents (data taken from Fig. 2). The timings used for complete continental breakups are 112 MYA for (Africa + South America) vs. (Madagascar + Indo/Sri Lanka), 100 MYA for Africa vs. South America, and 85 MYA for Madagascar vs. Indo/Sri Lanka [50-52]. The solid line indicates a 1:1 relationship between paleontological and molecular time estimates.

Among the fossil data points, four data points in the Paleozoic show a fairly good 1:1 relationship, whereas other points mostly in the Mesozoic are considerably below the line of 1:1 relationship. This might mean that the Mesozoic fossils do not really represent the oldest fossil for the corresponding lineages whereas this is not the case for older Paleozoic lineages. This situation is somewhat reminiscent of the apparent relative paucity of Mesozoic fossil evidence of tetrapods (mammals and birds) [58].

Several papers have noticed that molecular time estimations are consistently older than paleontological ones [2,3,5-7,59]. Benton and Ayala [60] have pointed out four pervasive biases that make molecular dates too old: i) too old calibration dates based on previous molecular studies; ii) undetected fast-evolving genes; iii) ancestral polymorphism that is maintained through long evolutionary period; and iv) asymmetric distributions of estimated times, with a constrained younger end but an unconstrained older end (this is caused because rates of evolution are constrained to be nonnegative, but the rates are unbounded above zero).

The first factor is not the case for the present study, because we did not use the calibration dates based on previous molecular studies, but used only those based on fossil records. The third factor would be the case when the used genomic regions are under the long-term balancing selection, but no mitochondrial gene has been reported to be under such selection. Regarding the second and fourth factors, we believe that they are also not the case for this study, because we used mitogenomic sequence data excluding peculiarly rapid evolving region (e.g., the control region), and because each mitochondrial gene used here was tested to perform well for dating vertebrate (tetrapod) divergences [61]. According to Benton and Ayala [60], for reliable dating "careful choice of genes may be a more appropriate strategy (than the larger data strategy), with a focus on long and fast-evolving (yet alignable) sequences." Our present study based on nearly whole mitogenomic sequence data fairly accommodates such condition.

Improved dating of teleostean divergences

We then conducted the divergence time estimation using the Gondwanan vicariance assumption regarding cichlids as additional time constraints (Fig. 4). Compared to the results shown in Figure 2 (without the additional time constraints), the means of estimated divergence times at various nodes are similar or somewhat larger (= 18 million years; see Table 3). However, the 95% credibility ranges of the estimated times overlap well between the two results, and the differences in mean values are not large, compared to potential error ranges in other elements, such as stochastic errors in molecular evolution and errors in dating fossils.

Figure 4. Divergence times estimated from the partitioned Bayesian analysis using both paleontological time constraints (Table 2) and biogeographical assumptions for the divergences of continental cichlid groups. The added time constraints on cichlid divergences are as follows: 112 MYA (lower) and 145 MYA (upper) for (Africa + South America) vs. (Madagascar + Indo/Sri Lanka); 100 MYA (lower) and 120 MYA (upper) for Africa vs. South America; and 85 MYA (lower) and 95 MYA (upper) for Madagascar vs. Indo/Sri Lanka [50-52]. See Fig. 2 legend for other details.

The addition of the cichlid constraints appears to shorten the 95% credibility intervals of the time estimates, especially for divergences occurring within Acanthomorpha 100–200 MYA. For example, our Figure 2 and Yamanoue et al. [55] estimated the divergence time of torafugu (Tetraodontiformes) and medaka (Beloniformes) to be approximately 159 (136–183) MYA and 184 (154–221) MYA, respectively. The cichlid constraints considerably narrowed the 95% credibility interval to 176 (163–191) MYA (Table 3), and also increased the precision of time estimates for other nodes. The use of ample molecular data from mitogenomic sequences also helped to narrow the credibility interval. For example, Kumazawa et al. [5] used two mitochondrial genes (NADH dehydrogenase subunit 2 and cytochrome b) and estimated the divergence between torafugu and zebrafish at 284 ± 28 (mean ± standard deviation) MYA, whereas our whole mitogenomic data set showed the divergence at 288 (268–307) MYA (Table 3).

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