Mitochondrial 16S rDNA Sequences Research Articles

Keratitis by Acanthamoeba triangularis: Report of Cases and Characterization of Isolates

Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.

Molecular phylogenetic analyses of mullets (Mugilidae, Mugiliformes) based on two mitochondrial genes

Escuela de Ciencias Aplicadas del Mar, Universidad de Oriente, Isla de Margarita,VenezuelaSummaryMolecular phylogenetic studies are very scarce for the Mugil-idae family; the present analysis using DNA sequences of themitochondrial 16S rRNA and cytochrome b genes is the firststudy involving Brazilian mugilids. The results corroborate themonophyly of Mugil and are elucidative for the taxonomy ofBrazilian mugilids. Mugil curema is clearly divided into twogenetically distinct taxa, M. curema type I being closer relatedto Mugil hospes and the true M. curema (type II) groupingsignificantly with M. incilis. The results also suggest that Mugilliza and Mugil platanus should be treated as a single species oreven populations of Mugil cephalus.IntroductionMugilids are coastal marine, brackish water and freshwaterfishes, distributed worldwide in tropical and temperate seas,migrating offshore to spawn. Mullets are very importantcommercial species and are highly exploited throughout theirdistribution (Cervigo´n, 1993; Cervigo´n et al., 1993). They arepopularly known in Brazil as tainha, parati, curima˜, cai´ca andpratiqueira (Menezes, 1983; Menezes and Figueiredo, 1985).The most recent taxonomic revision (Thomson, 1997) recog-nizes 14 genera and 64 valid species, Mugil being the only oneoccurring in the Atlantic coastal waters of Brazil. The numberof mugilid species reported for the northern coast of SouthAmerica, including the entire Brazilian coast, varies between 7and 8 (Cervigo´n et al., 1993; Thomson, 1997; Menezes et al.,2003). The discrepancies are related to Mugil hospes,M. cephalus, M. platanus and the suppressed name ofM. gaimardianus.Accurate species identification is the first step in eachsuccessful conservation and fisheries management program(Ludwig, 2006). Identification of mugilid species is problem-atic, especially in juvenile stages, because of their highlyconserved external morphology and the paucity of usefultaxonomic characters (Menezes, 1983; Menezes and Figuei-redo, 1985; Cervigo´n, 1993; Thomson, 1997) and homo-geneous karyological structure. The majority of species showsa diploid number of 48 chromosomes (Cataudella et al., 1974;Delgado et al., 1992; Jorda˜o et al., 1992; Crosetti et al., 1993;Rossi et al., 1996, 1997; Nirchio et al., 2001, 2003), except forM. curema which has a diploid number of 24 or 28 (Le GrandeandFitzsimons,1976;Nirchio et al.,2003,2005),andM.incilispresenting 2n = 28 (Galetti et al., 2000).Because of the economic importance of this fish family it isessential to improve the current identification keys and takeadvantage of the powerful molecular markers now available.Mitochondrial DNA has been widely used in fish taxonomybut only a few Mediterranean mugilid taxa have so far beensubjected to molecular phylogenetic analysis. Caldara et al.(1996) used the mitochondrial cytochrome b and 12S rRNAgenes to evaluate the phylogenetic relationships among Chelon,Oedalechilus, Liza and Mugil. According to these authors,based on DNA sequences the arrangement produced was verydifferent from the current taxonomy based on morphology.Nuclear data of allozymes and mitochondrial 16S rDNAsequences (Rossi et al., 2004) suggest that the Mediterraneanspecies of Liza do not form a monophyletic group, inagreement with the previous results of Caldara et al. (1996).The present phylogenetic analysis based on the 16S rRNAand cytochrome b mitochondrial genes is the first for Brazilianmugilids (Western Atlantic), and includes previously studiedMediterranean taxa (Mugil, Chelon and Oedalechilus). Thephylogenetic trees depicted from both genes are congruent andsurprisingly reveal two possible species morphologically iden-tified as Mugil curema, but clearly distinguished by the DNAanalyses. Molecular data also suggest that Mugil liza andMugil platanus should be treated as a single species or possiblypopulations of Mugil cephalus.Materials and methods

Monophyly and phylogenetic relationships of Thereva and therevine genus-groups (Insecta:Diptera:Therevidae) based on EF-1α, 28S rDNA and mitochondrial 16S rDNA sequences

Phylogenetic analyses using 28S rDNA, elongation factor (EF)-1α, and mt 16S rDNA sequences were performed to test the monophyly of Thereva Latreille. Two of the three Afrotropical Thereva species groups lack the genitalia characters that unambiguously diagnose Thereva in the Holarctic Region, but phylogenetic relationships among Thereva species groups and therevine genera are poorly understood. Using an extensive taxonomic sample (39 of the 62 therevine genera) and Thereva, sensu lato (15 spp.), simultaneous analyses of all three gene partitions recovered Nearctic and Palaearctic Thereva species in a well supported clade that includes the Afrotropical seminitida-group but excludes the Afrotropical analis- and turneri-groups. Stronger phylogenetic signal from the EF-1α partition, measured by the skewness statistic and proportion of total parsimony informative characters, dominated conflicting signal from the 16S partition and weaker, but more congruent, signal from 28S. Reducing the taxonomic sample in analyses of Therevinae reduced homoplasy, increased phylogenetic structure and partitioned Bremer support values and reduced incongruence with 28S for the 16S partition. Although molecular analyses yielded partial recovery of informal therevine genus-groups, morphological diagnoses of higher-level groups are poorly supported with the exception of Cyclotelini. The ‘Holarctic radiation’ refers to a diverse clade of genera closely related to Pandivirilia Irwin & Lyneborg and Acrosathe Irwin & Lyneborg widely distributed throughout the Holarctic Region that is the sister-group to Thereva, sensu stricto. Results from these analyses underscore the importance of male and female genitalia characters in recognising monophyletic groups and regional endemism in therevine diversification.

Cryptic diversity in Engaeus Erichson, Geocharax Clark and Gramastacus Riek (Decapoda : Parastacidae) revealed by mitochondrial 16S rDNA sequences

Nucleotide sequence data from the mitochondrial 16S rDNA region were utilised to investigate phylogenetic relationships and species boundaries among Australian freshwater crayfish belonging to the genera Engaeus Erichson, 1846, Geocharax Clark, 1936 and Gramastacus Riek, 1972. Geocharax and Gramastacus were found to be monophyletic genera but one species currently assigned to Engaeus may belong to another genus. Relationships between the three existing genera were not resolved. Analysis of species boundaries within Geocharax suggests that there are an additional two species in this genus, and our analysis of Gramastacus indicates that undescribed populations from central New South Wales may comprise a second species. The data provide at least one instance of a taxon crossing the Great Dividing Range and provide confirmation of previously proposed hypotheses seeking to explain trans-Bass Strait distributions of species.

Notes on New World <I>Persicargas</I> Ticks (Acari: Argasidae) with Description of Female <I>Argas</I> (<I>P</I>.) <I>keiransi</I>

The female of Argas (Persicargas) keiransi Estrada-Peña, Venzal & González-Acuña (Acari: Argasidae) is described from specimens collected in the type locality in Chile. The female has a unique combination of characters in the dorsal cuticle and in the number and pattern of ventrolateral setae in the basis capituli. Additional features on tarsus I chaetotaxy and dorsal plate are provided for the larva of the species, as observed on flat specimens derived from engorged females collected in the type locality. Keys for the larvae of the New World Persicargas are provided as well as illustrations for prominent characters in hypostome and posterolateral setae. The mitochondrial 16S rDNA sequence of A. keiransi has been obtained, and an analysis of the phylogenetic relationships with other ticks is included. Phylogenetic analysis provides support for a Persicargas grouping of species, separated from species in Argas.

AN EXTREMELY LOW GENETIC DIVERGENCE ACROSS THE RANGE OF EUSCORPIUS ITALICUS (SCORPIONES, EUSCORPIIDAE)

Abstract Little or no genetic divergence is detected using mitochondrial 16S rDNA sequence comparisons across the entire geographic range of the scorpion Euscorpius italicus (Herbst 1800) from Switzerland, Italy, Slovenia, Greece and Turkey. This is consistent with known absence of patterns of allozymes and morphological variation. Euscorpius italicus is found almost exclusively in human habitations. Its sister species, E. naupliensis, exhibits much higher genetic diversity within southern Greece. We suggest that the natural populations of the thermophilic E. italicus underwent a bottleneck during the glaciations, and that its modern range could be a result of dispersal with humans.

Sister species within the Triops cancriformis lineage (Crustacea, Notostraca)

We investigated the phylogenetic relationships among the three presently recognized subspecies of the tadpole shrimp, Triops cancriformis, using mitochondrial 16S and 12S rDNA sequences. Our results indicate that the taxon is divided into two distinct lineages. One lineage is formed of T. c. cancriformis populations and samples from northern Spain that had been classified as T. c. simplex in the most recent literature. The second lineage comprises all populations of T. c. mauritanicus and northern African populations of T. c. simplex. These two main lineages separated 2.3 to 8.9 million years ago, based on the range of inferred molecular clocks recognized for crustacean mtDNA sequence divergence. Percentages of divergence are in the range reported for recognized species in other notostracan lineages and we therefore propose to recognize them as two species, Triops cancriformis and Triops mauritanicus. The latter would comprise two subspecies in northern Africa, one consisting of the Moroccan populations of the former T. c. mauritanicus, the other comprising the African populations of the former T. c. simplex. It also includes three as‐yet unnamed lineages. A comparison of morphological characters with the molecular data revealed that the former T. c. simplex cannot be reliably separated from T. c. cancriformis, using morphological characters that have hitherto been used to distinguish among subspecies of T. cancriformis. Our investigation is the first to demonstrate the presence of T. c. cancriformis in Africa (Tunisia). The genetic haplotypes of these populations are identical with haplotypes also occurring in Central and Western Europe, as well as in Sicily. Therefore, we hypothesize that the African populations of T. c. cancriformis represent a result of repeated long‐distance dispersal across the Mediterranean Sea.

Phylogenetic analysis of flatfish (Order Pleuronectiformes) based on mitochondrial 16s rDNA sequences

The phylogenetic relationships of the order Pleuronectiformes are controversial and at some crucial points remain unresolved. To date most phylogenetic studies on this order have been based on morpho-anatomical criteria, whereas only a few sequence comparisons based studies have been reported. In the present study, the phylogenetic relationships of 30 flatfish species pertaining to seven different families were examined by sequence analysis of the first half of the 16S mitochondrial DNA gene. The results obtained did not support percoids as the sister group of pleuronectiforms. The monophyletic origin of most families analyzed, Soleidae, Scophthalmidae, Achiridae, Pleuronectidae and Bothidae, was strongly supported, except for Paralichthyidae which was clearly subdivided into two groups, one of them associated with high confidence to Pleuronectidae. The analysis of the 16S rRNA gene also suggested the monophyly of Pleuronectiforms as the most probable hypothesis and consistently supported some major interfamily groupings.

Molecular phylogeny of some genera of Pamphagidae (Acridoidea, Orthoptera) from China based on mitochondrial 16S rDNA sequences

Based on the mitochondrial 16S ribosomal DNA partial sequences (473 bp) of 9 species of Pamphagidae (Acridoidea, Orthoptera) from China and of 4 species of Pamphagidae and 2 species of Pyrgomorphidae and Acrididae (as outgroups) retrieved from GenBank, we constructed the molecular phylogeny using the Neighbor Joining (NJ) and Minimum Evolution (ME) methods based on the nucleotide Kimura 2-parameter model. The results of our study shown that: 1) the ranges of the 16S rDNA nucleotide divergence between two species of a genus were 0.21%, among genera of a subfamily were 0.42–3.38%, and among subfamilies of Pamphagidae were 1.90–8.88%, respectively. The phylogenetic tree shows that: 1) all Pamphagidae taxa form a monophyletic clade, and are well separated from the outgroup; 2) the African taxa Porthetinae (Lobosceliana brevicornis) and Akicerinae (Batrachotetrix sp.) are distinctly separated from the Chinese taxa Prionotropisinae; 3) Haplotropis bruneriana and Glauia terrea of Pamphaginae are nested in the middle of the tree, but their phylogenetic status is uncertain in this study; 4) 8 genera of Asiotmethis, Beybienkia, Mongolotmethis, Sinotmethis, Rhinotmethis, Filchnerella, Eotmethis and Pseudotmethis from China are all grouped into the subfamily Prionotropisinae, but their phylogenetic relationships are not clearly resolved.

Phylogeny of Tubificidae (Annelida, Clitellata) based on mitochondrial and nuclear sequence data

The tubificid clitellates are a common component in the freshwater bottom fauna and are also the most abundant oligochaete group in marine habitats. There are over 800 described species classified in six subfamilies; Tubificinae, Limnodriloidinae, Rhyacodrilinae, Telmatodrilinae, Phallodrilinae, and Naidinae. In this study we examine the phylogenetic relationships in Tubificidae using a combination of mitochondrial 16S rDNA and nuclear 18S rDNA sequence data. Sequences were obtained from five outgroup and 56 ingroup taxa, including five of the six subfamilies of Tubificidae. The data were analysed by maximum parsimony and Bayesian inference. The resulting tree topologies are virtually without conflict. Several associations traditionally recognized within the family Tubificidae are supported, in the Bayesian analysis including a sister group relationship between Tubificinae and Limnodriloidinae. The results also indicate that Rhyacodrilinae is polyphyletic—some of its members ( Heterodrilus spp.) fall into a clade with Phallodrilinae, all other groups with Naidinae. Naidinae is also polyphyletic with two rhyacodriline genera, Monopylephorus and Ainudrilus, nested within. Most of the tubificid genera included in the study are supported as monophyletic; however, Tubifex and Limnodriloides are refuted, and Tubificoides is unresolved from other tubificine taxa.

Evaluation of taxonomic validity of four species of Acanthamoeba: A. divionensis, A. paradivionensis, A. mauritaniensis, and A. rhysodes, inferred from molecular analyses.

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.

Phylogeny and divergence time estimation of cheilostome bryozoans based on mitochodrial 16S rRNA sequences

The mitochondrial 16S rDNA sequences of 40 species of cheilostome bryozoans including those of 24 species newly determined were used to reconstruct the phylogenetic tree using neighboringjoining and maximumparsimony methods. By applying molecular clock technique on the basis of the appropriate phylogeny and the fossil record, the divergence times of the two main cheilostome groups, Anasca and Ascophora sensu stricto, were estimated. The results show that the molecular phylogeny of the higher taxonomic groups (superfamilies and higher taxa) of cheilostome bryozoans is mostly in conflict with the morphologybased phylogenetic trees; the divergence of the extant groups of Anasca and those of Ascophora sensu stricto is estimated to have happened about 263 Ma (Permian Guadalupian Epoch) and 183 Ma (Early Jurassic), respectively.

Novel conopeptides of the I-superfamily occur in several clades of cone snails

The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups.

Molecular and morphological data reveal cryptic taxonomic diversity in the terrestrial slug complex Arion subfuscus/fuscus (Mollusca, Pulmonata, Arionidae) in continental north-west Europe

The importance and abundance of cryptic species among invertebrate taxa is well documented. Nowadays, taxonomic, phylogenetic and conservation biological studies frequently use molecular markers to delineate cryptic taxa. Such studies, however, often face the problem of the differential resolution of the molecular markers and techniques involved. This issue is explored in the present study of cryptic taxa within the terrestrial slug complex Arion subfuscus/fuscus in continental north-west Europe. To this end, morphological, allozyme and mitochondrial 16S rDNA sequence data have been jointly evaluated. Using allozyme data and gonad type, two distinct groups were consistently delineated, even under sympatric conditions. The 16S rDNA data strongly supported both those groups and even suggested the presence of three distinct taxa within one of them. However, in view of: (1) the allopatric distribution of three OTUs, (2) the lack of allozyme or morphological differentiation, and (3) the extremely high degree of intraspecific mtDNA variation reported in pulmonate gastropods, they are, for the time being, not regarded as valid species under the biological species concept. By means of 16S rDNA and allozyme data, the position of type and topotype material of A. subfuscus s.s. and A. fuscus relative to the newly defined OTUs was determined, thus clarifying the nomenclature of this species complex. Additionally, gonad type proved to be a useful character for distinguishing the two species in north-west Europe.

First records of egg masses of Nototodarus gouldi McCoy, 1888 (Mollusca: Cephalopoda: Ommastrephidae), with comments on egg‐mass susceptibility to damage by fisheries trawl

The egg mass and embryos of the ommastrephid squid Nototodarus gouldi McCoy, 1888 are reported for the first time, their identity confirmed by mitochondrial 16S rDNA sequence determination. The egg mass is a free‐floating gelatinous sphere of at least 1.5 m diameter and contains an estimated several thousand randomly distributed eggs; similar egg masses recorded from north‐eastern New Zealand waters of 1.0–2.0 m diameter are reported. Observed fluctuations in populations for this and other squid species may be a partial result of trawl damage to the egg masses.

Phylogenetic position of Phthiraptera (Insecta: Paraneoptera) and elevated rate of evolution in mitochondrial 12S and 16S rDNA

Phthiraptera (chewing and sucking lice) and Psocoptera (booklice and barklice) are closely related to each other and compose the monophyletic taxon Psocodea. However, there are two hypotheses regarding their phylogenetic relationship: (1) monophyletic Psocoptera is the sister group of Phthiraptera or (2) Psocoptera is paraphyletic, and Liposcelididae of Psocoptera is the sister group of Phthiraptera. Each hypothesis is supported morphologically and/or embryologically, and this problem has not yet been resolved. In the present study, the phylogenetic position of Phthiraptera was examined using mitochondrial 12S and 16S rDNA sequences, with three methods of phylogenetic analysis. Results of all analyses strongly supported the close relationship between Phthiraptera and Liposcelididae. Results of the present analyses also provided some insight into the elevated rate of evolution in mitochondrial DNA (mtDNA) in Phthiraptera. An elevated substitution rate of mtDNA appears to originate in the common ancestor of Phthiraptera and Liposcelididae, and directly corresponds to an increased G + C content. Therefore, the elevated substitution rate of mtDNA in Phthiraptera and Liposcelididae appears to be directional. A high diversity of 12S rDNA secondary structure was also observed in wide range of Phthiraptera and Liposcelididae, but these structures seem to have evolved independently in different clades.

Evidence for survival of Pleistocene climatic changes in Northern refugia by the land snail Trochoidea geyeri (Soós 1926) (Helicellinae, Stylommatophora)

BackgroundThe study of organisms with restricted dispersal abilities and presence in the fossil record is particularly adequate to understand the impact of climate changes on the distribution and genetic structure of species. Trochoidea geyeri (Soós 1926) is a land snail restricted to a patchy, insular distribution in Germany and France. Fossil evidence suggests that current populations of T. geyeri are relicts of a much more widespread distribution during more favourable climatic periods in the Pleistocene.ResultsPhylogeographic analysis of the mitochondrial 16S rDNA and nuclear ITS-1 sequence variation was used to infer the history of the remnant populations of T. geyeri. Nested clade analysis for both loci suggested that the origin of the species is in the Provence from where it expanded its range first to Southwest France and subsequently from there to Germany. Estimated divergence times predating the last glacial maximum between 25–17 ka implied that the colonization of the northern part of the current species range occurred during the Pleistocene.ConclusionWe conclude that T. geyeri could quite successfully persist in cryptic refugia during major climatic changes in the past, despite of a restricted capacity of individuals to actively avoid unfavourable conditions.

Relationships and origin of endemic Lake Baikal gastropods (Caenogastropoda: Rissooidea) based on mitochondrial DNA sequences

The phylogenetic relationships and the origin of two groups of rissooid freshwater snails endemic to Lake Baikal were investigated using partial mitochondrial COI, 12S rDNA, and 16S rDNA sequences. The Baikalian Benedictiinae proved to be closely related to the Lithoglyphinae. According to a molecular clock estimate the two groups diverged in the Paleogene. The Benedictiinae might have evolved autochthonously in precursors of Lake Baikal. The Baikalian Baicaliidae are probably most closely related to the Amnicolidae and the Bithyniidae. These groups diverged at the latest during the Cretaceous. Thus the origin of the Baicaliidae predates the origin of the Baikal rift zone. The Baicaliidae evolved probably in other Central Asian freshwater reservoirs. However, the radiation of the extant Baicaliidae only started in the Neogene and might have occurred autochthonously in Lake Baikal. The conchological similarity of the Baicaliidae and the Pyrgulidae is due to convergence. The Pyrgulidae diverged from the common stem lineage of the other hydrobiid families at the latest in the Jurassic. The Bithyniidae is derived from hydrobiids and is related to the Amnicolidae.

Recent origin of sub-Antarctic notothenioids

Comparison of partial mitochondrial 12S and 16S rDNA sequences from non-Antarctic notothenioid fishes – an icefish Champsocephalus esox and two members of the genus Patagonotothen – and their sister species from the Southern Ocean suggests that their divergence took place 1.7 and 6.6–7 million years ago, respectively, i.e. much later than the formation of the Antarctic Polar Front (20–25 million years ago).

Ancient tetraploidy and slow molecular evolution in Scaphiophryne: ecological correlates of speciation mode in Malagasy relict amphibians.

Karyotypes of three microhylid frog species of the Malagasy relict genus Scaphiophryne were studied: Scaphiophryne gottlebei, S. madagascariensis and S. spinosa. The latter two showed a plesiomorphic ranoid karyotype of 2n = 26. In contrast, tetraploidy was demonstrated in S. gottlebei, which constitutes an exceptional state among Malagasy amphibians. A combination of different banding techniques and of rDNA-FISH provided evidence for allopolyploidy in the species and for a completed subsequent functional and structural diploidization. Phylogenetic analysis of mitochondrial 16S rDNA sequences revealed a significant deceleration of nucleotide substitution rates in Scaphiophryne. The tetraploidy of S. gottlebei probably occurred early in their radiation. Ecological and behavioural patterns of Scaphiophryne probably favoured intraspecific gene flow and hybridization events, thereby leading to slow molecular substitution rates and to allopolyploid chromosome speciation in S. gottlebei.