Molecular evolution of the primate developmental genes MSX1 and PAX9

In primates, the craniofacial skeleton and the dentition are marked by high levels of interspecific variation. Despite this, there are few comparative species studies conducted at the molecular level to investigate this functional diversity. We have determined nucleotide sequences of MSX1 and PAX9, two developmental genes, in a sample of 27 diverse primate species in order to identify coding or regulatory variation that may be associated with phenotypic diversity. Our analyses have identified four highly conserved noncoding sequences, including one that is conserved across primates and with dogs but not with mice. Although we find that substitution rates vary significantly across MSX1 exons, comparisons of nonsynonymous and synonymous substitution rates (dN/dS) suggest that, as a whole, MSX1 and PAX9 amino acid sequences have been under functional constraint throughout primate evolution. Compared to all other primates in our sample, our analysis of exon 1 in MSX1 finds an unusual pattern of amino acid substitution for Tarsius syrichta, a member of a lineage (tarsiers) that has many unique features among primates. For example, tarsiers are the only extant primates without deciduous incisors, and MSX1 is expressed exclusively in the incisor regions during the earliest stages of dental development. Our overall results provide insight into the utility of comparative species analyses of highly conserved developmental genes and their roles in the evolution of complex phenotypes.     

Multiple data sets, high homoplasy, and the phylogeny of softshell turtles (Testudines: Trionychidae)

We present a phylogenetic hypothesis and novel, rank-free classification for all extant species of softshell turtles (Testudines:Trionychidae). Our data set included DNA sequence data from two mitochondrial protein-coding genes and a approximately 1-kb nuclear intron for 23 of 26 recognized species, and 59 previously published morphological characters for a complimentary set of 24 species. The combined data set provided complete taxonomic coverage for this globally distributed clade of turtles, with incomplete data for a few taxa. Although our taxonomic sampling is complete, most of the modern taxa are representatives of old and very divergent lineages. Thus, due to biological realities, our sampling consists of one or a few representatives of several ancient lineages across a relatively deep phylogenetic tree. Our analyses of the combined data set converge on a set of well-supported relationships, which is in accord with many aspects of traditional softshell systematics including the monophyly of the Cyclanorbinae and Trionychinae. However, our results conflict with other aspects of current taxonomy and indicate that most of the currently recognized tribes are not monophyletic. We use this strong estimate of the phylogeny of softshell turtles for two purposes: (1) as the basis for a novel rank-free classification, and (2) to retrospectively examine strategies for analyzing highly homoplasious mtDNA data in deep phylogenetic problems where increased taxon sampling is not an option. Weeded and weighted parsimony, and model-based techniques, generally improved the phylogenetic performance of highly homoplasious mtDNA sequences, but no single strategy completely mitigated the problems of associated with these highly homoplasious data. Many deep nodes in the softshell turtle phylogeny were confidently recovered only after the addition of largely nonhomoplasious data from the nuclear intron.     

Primate molecular divergence dates

With genomic data, alignments can be assembled that greatly increase the number of informative sites for analysis of molecular divergence dates. Here, we present an estimate of the molecular divergence dates for all of the major primate groups. These date estimates are based on a Bayesian analysis of approximately 59.8 kbp of genomic data from 13 primates and 6 mammalian outgroups, using a range of paleontologically supported calibration estimates. Results support a Cretaceous last common ancestor of extant primates (approximately 77 mya), an Eocene divergence between platyrrhine and catarrhine primates (approximately 43 mya), an Oligocene origin of apes and Old World monkeys (approximately 31 mya), and an early Miocene (approximately 18 mya) divergence of Asian and African great apes. These dates are examined in the context of other molecular clock studies.     

Phylogenetic patterns in populations of Chilean species of the genus Orestias (Teleostei: Cyprinodontidae): results of mitochondrial DNA analysis

Patterns of molecular genetic differentiation among taxa of the "agassii species complex" (Parenti, 1984) were analysed based on partial mtDNA control region sequences. Special attention has been paid to Chilean populations of Orestias agassii and species from isolated lakes of northern Chile, e.g., O. agassii, Orestias chungarensis, Orestias parinacotensis, Orestias laucaensis, and Orestias ascotanensis. Orestias tschudii, Orestias luteus, and Orestias ispi were analysed comparatively. Our findings support the utility of mtDNA control region sequences for phylogenetic studies within the "agassii species complex" and confirmed the monophyly of this particular lineage, excluding O. luteus. However, the monophyly of further morphologically defined lineages within the "agassii complex" appears doubtful. No support was found for the utility of these data sets for inferring phylogenetic relationships between more distantly related taxa originating from Lake Titicaca.     

Complete Mitochondrial Genomes and Eutherian Evolution

Recent large-scale nuclear DNA phylogenies have supported unconventional interordinal relationships among modern eutherians as well as divergence dates (100 mya) that substantially predate the first appearance of fossils from modern eutherians near the Cretaceous/Cenozoic (K/T) boundary (65-70 mya). For comparison to the nuclear data, I analyzed 12 complete mitochondrial DNA (mtDNA) protein-coding genes (10,677 bp) from 53 eutherian taxa, using maximum-likelihood methods to estimate model parameters (GTR + I + ) and to optimize topology and branch-length estimates. Although closely resembling the nuclear DNA trees, the mtDNA maximum-likelihood tree is just one of seven statistically indistinguishable ( lnL 1.747) trees, each suggesting different evolutionary relationships. This 53-taxon data set and another including 56 taxa provide no statistically significant support for a monophyletic afrotherian clade. In fact, these mitochondrial DNA sequences fail to support the monophyly of three putative eutherian divisions suggested by the nuclear data (Afrotheria, Laurasiatheria or Euarchontoglires). By comparison to well-supported branches describing relationships among families, those describing interordinal relationships are extremely short and only tenuously supported. Neither these sequences, nor sequences simulated under a known tree, fully resolve any interordinal relationship. Even simulated sequences that are twice as long (22kb) as mtDNA protein-coding genes are too short and too saturated to resolve the deepest and shortest interordinal relationships. Further, the mammalian mtDNA sequences appear to depart significantly from molecular-clock and quartet dating assumptions. Unlike recent nuclear DNA studies, I find that mtDNA genes, by themselves, are inadequate to describe relationships or divergence times at the base of the eutherian tree.     

Evolutionary history of the genus Rhamdia (Teleostei: Pimelodidae) in Central America

We constructed phylogenetic hypotheses for Mesoamerican Rhamdia, the only genus of primary freshwater fish represented by sympatric species across Central America. Phylogenetic relationships were inferred from analysis of 1990 base pairs (bp) of mitochondrial DNA (mtDNA), represented by the complete nucleotide sequences of the cytochrome b (cyt b) and the ATP synthase 8 and 6 (ATPase 8/6) genes. We sequenced 120 individuals from 53 drainages to provide a comprehensive geographic picture of Central American Rhamdia systematics and phylogeography. Phylogeographic analysis distinguished multiple Rhamdia mtDNA lineages, and the geographic congruence across evolutionarily independent Rhamdia clades indicated that vicariance has played a strong role in the Mesoamerican diversification of this genus. Phylogenetic analyses of species-level relationships provide strong support for the monophyly of a trans-Andean clade of three evolutionarily equivalent Rhamdia taxa: R. guatemalensis, R. laticauda, and R. cinerascens. Application of fish-based mitochondrial DNA clocks ticking at 1.3-1.5% sequence divergence per million years (Ma), suggests that the split between cis- and trans-Andean Rhamdia extends back about 8 Ma, and the three distinct trans-Andean Rhamdia clades split about 6 Ma ago. Thus the mtDNA divergence observed between cis- and trans-Andean Rhamdia species is too low to support an ancient colonization of Central America in the Late Cretaceous or Paleocene as had been hypothesized in one colonization model for Mesoamerican fishes. Rather the mtDNA data indicate that Rhamdia most likely colonized Central America in the late Miocene or Pliocene, promoting a strong role for the Isthmus of Panamá in the Mesoamerican expansion of this genus. Basal polytomies suggest that both the R. laticauda and R. guatemalensis clades spread rapidly across the Central American landscape, but differences in the average mtDNA genetic distances among clades comprising the two species, indicate that the R. laticauda spread and diversified across Mesoamerica about 1 million years before R. guatemalensis.     

Molecular phylogeny of fire ants of the Solenopsis saevissima species-group based on mtDNA sequences

The systematics of South American fire ants (Solenopsis saevissima species-group) has been plagued by difficulties in recognizing species and their relationships on the basis of morphological characters. We surveyed mtDNA sequences from 623 individuals representing 13 described and undescribed species within the species-group and 18 individuals representing other major Solenopsis lineages to generate a phylogeny of the mitochondrial genome. Our analyses support the monophyly of the S. saevissima species-group, consistent with a single Neotropical origin and radiation of this important group of ants, as well as the monophyly of the socially polymorphic species within the group, consistent with a single origin of polygyny (multiple queens per colony) as a derived form of social organization. The mtDNA sequences of the inquiline social parasite S. daguerrei form a clade that appears to be distantly related to sequences from the several host species, consistent with the view that advanced social parasitism did not evolve via sympatric speciation of intraspecific parasites. An important general finding is that species-level polyphyly of the mtDNA appears to be the rule in this group of ants. The existence of multiple divergent mtDNA lineages within several nominal species (including the pest S. invicta) suggests that the pattern of widespread polyphyly often stems from morphological delimitation that overcircumscribes species. However, in two cases the mtDNA polyphyly likely results from recent interspecific hybridization. While resolving species boundaries and relationships is important for understanding general patterns of diversification of South American fire ants, these issues are of added importance because invasive fire ants are emerging as global pests and becoming important model organisms for evolutionary research.     

Molecular phylogeny and divergence time estimates for major rodent groups: evidence from multiple genes

The order Rodentia contains half of all extant mammal species, and from an evolutionary standpoint, there are persistent controversies surrounding the monophyly of the order, divergence dates for major lineages, and relationships among families. Exons of growth hormone receptor (GHR) and breast cancer susceptibility (BRCA1) genes were sequenced for a wide diversity of rodents and other mammals and combined with sequences of the mitochondrial 12S rRNA gene and previously published sequences of von Willebrand factor (vWF). Rodents exhibit rates of amino acid replacement twice those observed for nonrodents, and this rapid rate of evolution influences estimates of divergence dates. Based on GHR sequences, monophyly is supported, with the estimated divergence between hystricognaths and most sciurognaths dating to about 75 MYA. Most estimated dates of divergence are consistent with the fossil record, including a date of 23 MYA for Mus-Rattus divergence. These dates are considerably later than those derived from some other molecular studies. Among combined and separate analyses of the various gene sequences, moderate to strong support was found for several clades. GHR appears to have greater resolving power than do 12S or vWF. Despite its complete unresponsiveness to growth hormone, Cavia (and other hystricognaths) exhibits a conservative rate of change in the intracellular domain of GHR.     

Mitochondrial phylogenetics and evolution of mysticete whales

The phylogenetic relationships among baleen whales (Order: Cetacea) remain uncertain despite extensive research in cetacean molecular phylogenetics and a potential morphological sample size of over 2 million animals harvested. Questions remain regarding the number of species and the monophyly of genera, as well as higher order relationships. Here, we approach mysticete phylogeny with complete mitochondrial genome sequence analysis. We determined complete mtDNA sequences of 10 extant Mysticeti species, inferred their phylogenetic relationships, and estimated node divergence times. The mtDNA sequence analysis concurs with previous molecular studies in the ordering of the principal branches, with Balaenidae (right whales) as sister to all other mysticetes base, followed by Neobalaenidae (pygmy right whale), Eschrichtiidae (gray whale), and finally Balaenopteridae (rorquals + humpback whale). The mtDNA analysis further suggests that four lineages exist within the clade of Eschrichtiidae + Balaenopteridae, including a sister relationship between the humpback and fin whales, and a monophyletic group formed by the blue, sei, and Bryde's whales, each of which represents a newly recognized phylogenetic relationship in Mysticeti. We also estimated the divergence times of all extant mysticete species, accounting for evolutionary rate heterogeneity among lineages. When the mtDNA divergence estimates are compared with the mysticete fossil record, several lineages have molecular divergence estimates strikingly older than indicated by paleontological data. We suggest this discrepancy reflects both a large amount of ancestral polymorphism and long generation times of ancestral baleen whale populations.     

The whole mitochondrial genome sequence of the zebra finch (Taeniopygia guttata)

Here we describe the complete nucleotide sequence of the mitochondrial genome (16 583/4 bp) of the zebra finch (Taeniopygia guttata). Primers were designed based on highly conserved regions of an alignment of three passerine complete mitochondrial DNA (mtDNA) sequences. A combination of overlapping long polymerase chain reaction (PCR) purification, followed by fully nested PCR and sequencing was used to determine the complete mtDNA genome. Six birds, from distinct maternal lineages of a pedigreed population were sequenced. Five novel haplotypes were identified. These sequences provide the first data for sequence variation across the whole mitochondrial genome of a passerine bird species.     

The complete mitochondrial genome sequences of <Emphasis Type="Italic">Chelodina rugosa</Emphasis> and <Emphasis Type="Italic">Chelus fimbriata</Emphasis> (Pleurodira: Chelidae): implications of a common absence of initiation sites (O<Subscript>L</Subscript>) in pleurodiran turtles

Within the order Testudines, while phylogenetic analyses have been performed on the suborder Cryptodira with complete mitochondrial genomes (mitogenomes), mitogenomic information from another important suborder Pleurodira has been inadequate. In the present study, complete mitochondrial DNA (mtDNA) sequences of two chelid turtles Chelodina rugosa and Chelus fimbriata were firstly determined, the lengths of which were 16,582 and 16,661 bp respectively. As the typical vertebrate mitogenome, both mtDNAs consist of 13 protein coding genes, 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a long noncoding region (control region, CR). However, the initiation sites for light-strand replication (O_L), which has been identified in all reported Cryptodire mitogenomes, were not found in the putative position of the two chelid turtles and African helmeted turtle Pelomedusa subrufa. The results suggested that the absence of mitogenomic initiation sites (O_L) could be a characteristic of Pleurodira. Phylogenetic relationships of chelid turtles and other turtles were reconstructed using the reported mitogenomes. Both maximum likelihood (ML) and Bayesian inference (BI) analyses suggested the monophyly of Pleurodira and Cryptodira as well as a sister group relationship between the two chelid turtles with strong statistical support. This phylogenetic framework was also utilized to estimate divergence dates among lineages using relaxed-clock methods combined with fossil evidence. Divergence estimates revealed that genus Chelodina diverged from genus Chelus in Late Cretaceous (~83 million years ago (mya)), and the time is consistent with the vicariance of the fragments which was caused by Gondwana split.     

Are Guinea Pigs Rodents? The Importance of Adequate Models in Molecular Phylogenetics

The monophyly of Rodentia has repeatedly been challenged based on several studies of molecular sequence data. Most recently, D'Erchia et al. (1996) analyzed complete mtDNA sequences of 16 mammals and concluded that rodents are not monophyletic. We have reanalyzed these data using maximum-likelihood methods. We use two methods to test for significance of differences among alternative topologies and show that (1) models that incorporate variation in evolutionary rates across sites fit the data dramatically better than models used in the original analyses, (2) the mtDNA data fail to refute rodent monophyly, and (3) the original interpretation of strong support for nonmonophyly results from systematic error associated with an oversimplified model of sequence evolution. These analyses illustrate the importance of incorporating recent theoretical advances into molecular phylogenetic analyses, especially when results of these analyses conflict with classical hypotheses of relationships.     

Congruent Avian Phylogenies Inferred from Mitochondrial and Nuclear DNA Sequences

Recent molecular studies addressing the phylogenetic relationships of avian orders have had conflicting results. While studies using nuclear DNA sequences tend to support traditional taxonomic views, also supported by morphological data [(paleognaths (galloanseres (all other birds)))], with songbirds forming a clade within Neoaves (all other birds), analyses with complete mtDNA genomes have resulted in topologies that place songbirds as one of the earliest-diverging avian lineages. Considering that over half of the extant bird species are songbirds, these different results have very different implications for our understanding of avian evolution. We analyzed data sets comprising nearly 4 kb of mitochondrial DNA (mtDNA) (complete 12S, ND1, ND2, and cytochrome b) plus 600 bp of the nuclear gene c-mos for 15 birds that were chosen to represent all major avian clades and to minimize potential long-branch attraction problems; we used a partition-specific maximum likelihood approach. Our results show congruence with respect to the ingroup among phylogenies obtained with mtDNA and the nuclear gene c-mos, separately or combined. The data sets support a traditional avian taxonomy, with paleognaths (ratites and tinamous) occupying a basal position and with songbirds more derived and forming a monophyletic group. We also show that, for mtDNA studies, turtles may be a better outgroup for birds than crocodilians because of their slower rate of sequence evolution.     

A mitogenomic perspective on the basal teleostean phylogeny: resolving higher-level relationships with longer DNA sequences

A recent study demonstrated that mitochondrial genomic (mitogenomic) data comprising nucleotide sequences from the concatenated protein-coding (no 3rd codon positions) plus transfer RNA (stem regions only) genes reproduced the expected phylogeny of teleosts with high statistical support. We reexamined the interrelationships of the five major, basal teleostean lineages (Osteoglossomorpha, Elopomorpha, Clupeomorpha, Ostariophysi, and Protacanthopterygii; given various rankings) using mitogenomic data for which five alternative phylogenetic hypotheses have been previously proposed on the basis of both morphological and molecular analyses. In addition to previously determined complete mitochondrial DNA (mtDNA) sequences from eight basal teleosts and two outgroups, we determined the complete mtDNA sequences (excluding a portion of the control region) for two, purposefully chosen species of Osteoglossomorpha (Osteoglossum bicirrhosum and Pantodon buchholzi), and the data were subjected to maximumparsimony and maximum-likelihood analyses. The resultant tree topologies from the two methods were congruent, although they differed from any of the previously proposed hypotheses. Furthermore, the mitogenomic data confidently rejected all of these hypotheses with high statistical significance.     

Proboscidean mitogenomics: chronology and mode of elephant evolution using mastodon as outgroup

We have sequenced the complete mitochondrial genome of the extinct American mastodon (Mammut americanum) from an Alaskan fossil that is between 50,000 and 130,000 y old, extending the age range of genomic analyses by almost a complete glacial cycle. The sequence we obtained is substantially different from previously reported partial mastodon mitochondrial DNA sequences. By comparing those partial sequences to other proboscidean sequences, we conclude that we have obtained the first sequence of mastodon DNA ever reported. Using the sequence of the mastodon, which diverged 24–28 million years ago (mya) from the Elephantidae lineage, as an outgroup, we infer that the ancestors of African elephants diverged from the lineage leading to mammoths and Asian elephants approximately 7.6 mya and that mammoths and Asian elephants diverged approximately 6.7 mya. We also conclude that the nuclear genomes of the African savannah and forest elephants diverged approximately 4.0 mya, supporting the view that these two groups represent different species. Finally, we found the mitochondrial mutation rate of proboscideans to be roughly half of the rate in primates during at least the last 24 million years.     

The complete mitochondrial genome sequence of Oncicola luehei (Acanthocephala: Archiacanthocephala) and its phylogenetic position within Syndermata

In the present study, we determined the complete mitochondrial genome sequence of Oncicola luehei (14,281bp), the first archiacanthocephalan representative and the second complete sequence from the phylum Acanthocephala. The complete genome contains 36 genes including 12 protein coding genes, 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (rrnL and rrnS) as reported for other syndermatan species. All genes are encoded on the same strand. The overall nucleotide composition of O. luehei mtDNA is 37.7% T, 29.6% G, 22.5% A, and 10.2% C. The overall A+T content (60.2%) is much lower, compared to other syndermatan species reported so far, due to the high frequency (18.3%) of valine encoded by GTN in its protein-coding genes. Results from phylogenetic analyses of amino acid sequences for 10 protein-coding genes from 41 representatives of major metazoan groups including O. luehei supported monophyly of the phylum Acanthocephala and of the clade Syndermata (Acanthocephala+Rotifera), and the paraphyly of the clade Eurotatoria (classes Bdelloidea+Monogononta from phylum Rotifera). Considering the position of the acanthocephalan species within Syndermata, it is inferred that obligatory parasitism characteristic of acanthocephalans was acquired after the common ancestor of acanthocephalans diverged from its sister group, Bdelloidea. Additional comparison of complete mtDNA sequences from unsampled acanthocephalan lineages, especially classes Polyacanthocephala and Eoacanthocephala, is required to test if mtDNA provides reliable information for the evolutionary relationships and pattern of life history diversification found in the syndermatan groups.     

Higher-level systematics of rodents and divergence time estimates based on two congruent nuclear genes

Phylogenetic analysis of over 4600 aligned nucleotide sequences from two nuclear genes, growth hormone receptor and BRCA1, provided congruent phylogenies depicting relationships among the major lineages of rodents. Separate and combined analyses resulted in five major conclusions: (1) strong support for a monophyletic Myodonta (containing the superfamilies Muroidea + Dipodoidea), with subfamily Gerbillinae being more closely related to Murinae than is Sigmodontinae; (2) a sister-group relationship between the family Castoridae and the superfamily Geomyoidea; (3) monophyly of Ctenohystrica (containing the suborders Sciuravida and Hystricognatha); (4) a near polytomy among Myodonta (suborder Myomorpha), Pedetes (family Pedetidae, suborder Anomaluromorpha), Castoridae (suborder Sciuromorpha) + Geomyoidea (suborder Myomorpha), and Ctenohystrica; and (5) basal position of a monophyletic group containing Graphiurus (family Gliridae, suborder Myomorpha) + two members of the Sciuromorpha (Sciuridae + Aplodontidae). Divergence dates among rodents and primates were also estimated using the combined data. Applying a global molecular clock and a primate calibration point, divergence dates among rodents exceeded fossil-based dates but were generally compatible with other molecule-based dates estimated under similar conditions. However, when a relaxed molecular clock was applied, estimated divergence dates were highly compatible with the fossil record.     

Pleistocene effects on the European freshwater fish fauna: double origin of the cobitid genus Sabanejewia in the Danube basin (Osteichthyes: Cobitidae)

Biogeographical hypotheses of European freshwater fishes were inferred using phylogeographic analysis of the complete cytochrome b and ATP synthase 8 and 6 mitochondrial genes (1982bp). To test the relative importance of drainage origin versus Pleistocene glaciations in the origin of primary freshwater fishes in Europe, we reconstructed the phylogenetic relationships of the genus Sabanejewia which is distributed in European waters. The phylogenetic relationships recovered for the genus Sabanejewia (n=75) provide support for the monophyly of six main evolutionary mtDNA lineages: Sabanejewia larvata, Sabanejewia romanica, Sabanejewia aurata/Sabanejewia caucasica, Sabanejewia kubanica, Sabanejewia baltica, and the Danubian-Balkanian complex. The Caucasian-Caspian mtDNA lineages, S. kubanica, S. aurata/S. caucasica, and the Northern European S. baltica represents the sister group of the Danubian-Balkanian complex mtDNAclade, supporting a Caucasian-Northern European origin of most of mtDNA lineages of the Central European freshwater fish fauna. The mtDNA divergence observed between the Danubian Sabanejewia species is too dissimilar to support their contemporary origin. Rather, the mtDNA data suggest that the Danubian Sabanejewia lineages most likely have a double origin, indicating that the European Sabanejewia lineages have experienced different historical processes for the following reasons. First, the origin of the S. larvata and S. romanica mtDNA clades predates the origin of the Danubian-Balkanian complex, and our results showed that the completion of the Alps and the origin of the Danube drainage seem to have promoted the speciation of the earliest Sabanejewia clades in the Miocene. Second, small genetic distances and the geographical pattern found within the Danubian-Balkanian complex clade indicate that the lineages included in this clade spread recently across the Danube and Greek river drainages. The inclusion of the S. balcanica species within all mtDNA lineages suggests that cyclical cold periods during the Pleistocene glaciations have favoured its rapid expansion and genetic homogenisation across Central European and Greek waters.     

Evolution and dispersal of three closely related macaque species, Macaca mulatta, M. cyclopis, and M. fuscata, in the eastern Asia

Macaca mulatta, M. cyclopis and M. fuscata are three closely related species in the fascicularis species group. M. mulatta is wide-spread in Asia, while M. cyclopis and M. fuscata are restricted to Taiwan and Japan, respectively. Both M. cyclopis and M. fuscata are thought to be derived from ancient 'mulatta' populations in the eastern Asia. In this study, we analyzed sequences of mitochondrial DNA control region to provide genetic evidence for the evolution and dispersal scenario of the three species proposed by Fooden and Albrecht [Fooden, J., Albrecht, G.H. 1999. Tail-length evolution in fascicularis-group macaques (Cercopithecidae: Macaca). Int. J. Primatol. 20, 431-440]. Our results indicated that several localities in the southern China and Vietnam harbored multiple divergent mtDNA lineages that may not have evolved sympatrically. These divergent mtDNA lineages may have originated from different ancient northern populations that retreated into southern localities during glacial periods. However, the age of the southward retreat and the northward recolonization may be dated back to a more ancient past during late middle Pleistocene (0.12-0.18 mya) instead of during the LGM (0.018 mya). Times of gene divergence between M. mulatta and the two island species, estimated by mean nucleotide difference, suggest the ancestral populations colonized Taiwan and Japan around 0.38-0.44 mya. In addition, a more recent age of mulatta-cyclopis-fuscata population divergence (when ancient populations were isolated), estimated to be 0.17 mya by net nucleotide divergence, is suggested.     

AFLPs resolve cytonuclear discordance and increase resolution among barcheek darters (Percidae: Etheostoma: Catonotus)

The use of mitochondrial DNA (mtDNA) sequences in phylogenetic analysis has been the subject of increasing scrutiny. A recent phylogenetic study of barcheek darters (Percidae: Etheostoma: Catonotus) revealed cytonuclear discordance, discordance among mtDNA loci, and discordance between mtDNA and morphometric hypotheses. In particular, mtDNA analyses hypothesized a paraphyletic barcheek darter clade, and a combined mtDNA and nuclear sequence topology was not well resolved. Here, we used amplified fragment length polymorphisms to test the monophyly of barcheek darters and to resolve relationships within the group. By including multiple populations of each species, we were able to generate a highly resolved tree that supports both the monophyly of barcheek darters as well as recently elevated species within the group. Analysis of three mtDNA loci indicates that saturation of highly variable sites best explains the discordant topologies among mtDNA partitions.