AFLPs resolve phylogeny and reveal mitochondrial introgression within a species flock of African electric fish (Mormyroidea: Teleostei)

Estimating species phylogeny from a single gene tree can be especially problematic for studies of species flocks in which diversification has been rapid. Here we compare a phylogenetic hypothesis derived from cytochrome b (cyt b) sequences with another based on amplified fragment length polymorphisms (AFLP) for 60 specimens of a monophyletic riverine species flock of mormyrid electric fishes collected in Gabon, west-central Africa. We analyze the aligned cyt b sequences by Wagner parsimony and AFLP data generated from 10 primer combinations using neighbor-joining from a Nei-Li distance matrix, Wagner parsimony, and Dollo parsimony. The different analysis methods yield AFLP tree topologies with few conflicting nodes. Recovered basal relationships in the group are similar between cyt b and AFLP analyses, but differ substantially at many of the more derived nodes. More of the clades recovered with the AFLP characters are consistent with the morphological characters used to designate operational taxonomic units in this group. These results support our hypothesis that the mitochondrial gene tree differs from the overall species phylogeny due at least in part to mitochondrial introgession among lineages. Mapping the two forms of electric organ found in this group onto the AFLP tree suggests that posteriorly innervated electrocytes with nonpenetrating stalks have independently evolved from anteriorly innervated, penetrating-stalk electrocytes at least three times.     

Genetic structure of Eurasian and North American Leymus (Triticeae) wildryes assessed by chloroplast DNA sequences and AFLP profiles

Leymus is a genomically defined allopolyploid of genus Triticeae with two distinct subgenomes. Chloroplast DNA sequences of Eurasian and North American species are distinct and polyphyletic. However, phylogenies derived from chloroplast and nuclear DNA sequences are confounded by polyploidy and lack of polymorphism among many taxa. The AFLP technique can resolve phylogenetic relationships between closely related species, with a curvilinear relationship expected between the proportion of shared bands and nucleotide substitution rate (D), up to about 0.100 D. The objective of this study was to compare D and phylogenetic relationships among 16 Leymus taxa, based on chloroplast DNA sequences and multi-locus AFLP genotypes. Estimates of chloroplast D between taxa were 0.002 and 0.013 within and among continental regions, respectively. Estimates of AFLP D between taxa were 0.076 and 0.093 compared within and between continental regions, respectively, versus 0.024 within taxa. Bayesian and neighbor-joining cluster analyses effectively separated all AFLP genotypes by species, but showed that North American L. ambiguus is a hybrid species with nearly equal contributions from sympatric L. cinereus and L. salinus taxa. Two hierarchical AFLP clades, containing six North American taxa and four Eurasian taxa, had more than 98% bootstrap confidence with 0.071 and 0.055 D among taxa. Three other Eurasian taxa clustered with 79% and 89% confidence, with up to 0.79 D between taxa. These estimates provide benchmarks for phylogenetic comparisons of AFLP profiles, but three taxa could not be reliably grouped, which may reflect concurrent radiation of multiple lineages or lack of homologous AFLP characters caused by a high D.     

Genetic relationships and species authentication of Boesenbergia (Zingiberaceae) in Thailand based on AFLP and SSCP analyses

Genetic relationships of 15 Boesenbergia species were examined using AFLP analysis. A total of 893 fragments were generated from six primer combinations; of these, 99.78% were polymorphic. The mean genetic distance between pairs of taxa ranged from 0.435 to 0.935. The neighbor-joining tree resolved investigated species into two separate lineages (corresponding to species possessing compact or elongated inflorescences) and suggests a rapid radiation in Boesenbergia. In addition, SSCP patterns of the partial psbA–trnH spacer were not overlapping between different species and therefore can be used for authentication of Boesenbergia species. Morphologically similar Boesenbergia longiflora and Boesenbergia sp., which showed three SNPs based on psbA–trnH polymorphism were successfully differentiated by SSCP. Moreover, Boesenbergia curtisii displayed intraspecific ecomorphological variation but exhibited the same SSCP pattern. Therefore, AFLP and SSCP are rapid and cost-effective methods for analysis of genetic similarity and species identification in Boesenbergia.     

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Using AFLP to resolve phylogenetic relationships in a morphologically diversified plant species complex when nuclear and chloroplast sequences fail to reveal variability

Inferring phylogenetic relationships among closely related plant species is often difficult due to the lack of molecular markers exhibiting enough nucleotide variability at this taxonomic level. Moreover, gene tree does not necessary represent the true species tree because of random sorting of polymorphic alleles in different lineages. A solution to these problems is to use many amplified fragment length polymorphisms (AFLP) distributed throughout the whole genome, to infer cladistic and phenetic among-species relationships. Phylogenetic relationships among interfertile species of Trollius L. (Ranunculaceae) were investigated using nuclear DNA (ITS1+5.8S rRNA+ITS2) and chloroplast DNA (trnL intron and trnL-trnF intergene spacer) sequences, and AFLP markers. ITS sequences were not informative at the intrageneric level, but confirmed the sister relationship between Trollius and Adonis genera, and provided new information on the phylogenetic relationships among five Ranunculaceae genera. Chloroplast DNA was more informative among Trollius species, but not consistent with the sections previously described. AFLP proved to be a powerful tool to resolve the complex genetic relationships between the morphological entities constituting the genus Trollius. Although as much as 76.1% of the total AFLP variability was found within a priori defined morphological groups, the remaining 23.9% variability differentiating groups was sufficient to generate congruent and robust cladistic and phenetic trees. Several morphological traits, independent from those used to define groups, were mapped onto the molecular phylogeny, and their evolution discussed in relation to the absence/presence of pollinator-seed parasite Chiastocheta flies.     

Original birthplace of cultivated common buckwheat inferred from genetic relationships among cultivated populations and natural populations of wild common buckwheat revealed by AFLP analysis

The genetic relationships among seven cultivated populations and eight natural populations of wild common buckwheat were analyzed using amplified fragment length polymorphism (AFLP). The genetic distance was estimated for each pair of the 15 populations based on the AFLP data, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the genetic distance. All the cultivated populations were grouped in a cluster. The natural populations were grouped into two clusters composed of (1) the Sanjiang group (three populations from eastern Tibet and one population from Adong village of Yunnan province) and (2) two populations from Yunnan province and two populations from Sichuan province. The Sanjiang group is more closely related to cultivated populations. These results indicate that the direct ancestor of common buckwheat was natural populations of wild common buckwheat from the Sanjiang area.     

Hybridization in the section Mentha (Lamiaceae) inferred from AFLP markers

The amplified fragment length polymorphism (AFLP) method was used to evaluate genetic diversity and to assess genetic relationships within the section Mentha in order to clarify the taxonomy of several interspecific mint hybrids with molecular markers. To this end, genetic diversity of 62 Mentha accessions from different geographic origins, representing five species and three hybrids, was assessed. Three EcoRI/MseI AFLP primer combinations generated an average of 40 AFLP markers per primer combination, ranging in size from 50 to 500 base pairs (bp). The percentage of markers polymorphic ranged from 50% to 60% across all accessions studied. According to phenetic and cladistic analysis, the 62 mint accessions were grouped into two major clusters. Principal coordinates analysis separated species into well-defined groups, and clear relationships between species and hybrids could be described. Our AFLP analysis supports taxonomic classification established among Mentha species by conventional (morphological, cytological, and chemical) methods. It allows the assessment of phenetic relationships between species and the hybrids M. spicata and M. × piperita, largely cultivated all over the world for their menthol source, and provides new insights into the subdivision of M. spicata, based for the first time on molecular markers.     

AFLP analysis of genetic relationships in the tribe Datureae (Solanaceae)

The AFLP technique was evaluated as a tool for assessing species relationships within the tribe Datureae and genetic distances were estimated for 47 accessions of over 12 species. The phenetic trees from various analyses of the AFLP data gave very high co-phenetic correlation values, and were found to be consistent with previous trees based on the analysis of different data types, in particular ITS-1 sequences, isozymes and morphology, carried out on the same accessions. These results indicated that the AFLP technique is both an efficient and effective tool for determining genetic relationships among taxa in the Solanaceae. A new classification is proposed for the tribe Datureae, which maintains the arborescent species as a separate genus, Brugmansia, and recognises three sections within the genus Datura; Stramonium, Dutra and Ceratocaulis. D. discolor, previously placed in section Dutra, was found to be intermediate between sections Dutra and Stramonium. Received: 11 January 1999 / Accepted: 1 February 1999     

GENETIC POLYMORPHISM IN THE MORPHOLOGICALLY REDUCED DWARF MISTLETOES (ARCEUTHOBIUM,VISCACEAE): AN ELECTROPHORETIC STUDY

Arceuthobium is a well defined genus of obligate parasites of conifers. Assessment of taxonomic and phylogenetic relationships in this group is difficult owing to morphological reductions and complex host relationships. In the present study, genetic relationships within and among 19 taxa were examined using starch gel electrophoresis of triploid seed endosperm tissue. Allelic frequency data for eleven polymorphic loci were derived from analysis of 40 natural populations collected from the U.S. and Mexico. The genus showed remarkably high levels of genetic diversity: averaged across the 19 taxa, 66.7% of the loci were polymorphic with an average of 2.23 alleles per locus. This level of polymorphism is approximately double the average value reported for many dicotyledons and stands in contrast to the overall uniform morphology of these parasites. Unweighted pair group cluster analysis (UPGMA) of genetic similarity measures was conducted for all sampled populations and the results compared to a phenetic system of classification for the genus. Similarities between the two studies include the recognition of two subgenera based upon the segregation of the verticillately branched Arceuthobium americanum from the remainder of the taxa examined. Analysis of isozyme data supported a grouping of six taxa: A. vaginatum ssp. cryptopodum, A. vaginatum ssp. durangense, A. gillii, A. rubrum, A. divaricatum, and A. douglasii. The placement of the latter three taxa in other groups by phenetic criteria provides evidence for molecular divergence not seen using morphological features. The Campylopodum group of taxa comprised eleven members that were linked at similarity values of 80% or greater. Populations were not unambiguously grouped according to species as defined by the phenetic study or at similarity levels comparable to other well defined species in this study. This group is either not reproductively isolated or molecular differentiation is cryptic due to rapid adaptive radiation onto numerous host tree species.     

Suitability of AFLP markers for the study of genetic relationships among Korean native dogs.

To determine the genetic relationships among domestic dog breeds, we performed both a sequence comparison of mitochondrial DNA (mtDNA) and an amplified fragment length polymorphisms (AFLP) analysis. Three of four regions of mtDNA, cytochrome b, cytochrome oxidase subunit II, and 16S rRNA genes were highly homogeneous among dog breeds, whereas the other region, the control region, showed relatively high polymorphisms with a maximum percentage difference of 3.18%. However, the control region showed extensive polymorphism even within breeds, and the relationship tree derived from the data could not clearly delimit distinct breeds. 19 EcoRI/MseI primer combinations were used to generate AFLP markers among 25 dogs from 11 breeds including three Korean native dogs. These amplification reactions allowed the detection of more than 1900 amplification products of which 408 were identified as polymorphic bands. Unrooted neighbor-joining tree based on dissimilarity values showed that the Korean native dogs were clustered together with the Asian dogs and that the Asian originated dogs were clustered separately from Western originated dogs. A consensus tree using parsimony method also showed Korean native dogs were grouped separately from the other dogs with moderate bootstrap values. Taken together, it is concluded that AFLP analysis is a more informative tool for revealing genetic relationships among dog breeds than mtDNA sequence comparison.     

Phylogenetic analysis of Petunia sensu Jussieu (Solanaceae) using chloroplast DNA RFLP

BACKGROUND AND AIMS: The phylogenetic relationships of Petunia sensu Jussieu (Petunia sensu Wijsman plus Calibrachoa) are unclear. This study aimed to resolve this uncertainty using molecular evidence. METHODS: Phylogenetic trees of 52 taxa of Petunia sensu Jussieu were constructed using restriction fragment length polymorphism (RFLP) of chloroplast DNA digested with 19 restriction enzymes and hybridized with 12 cloned Nicotiana chloroplast DNA fragments as probes. KEY RESULTS: In all, 89 phylogenetically informative RFLPs were detected and one 50 % majority consensus tree was obtained, using the maximum parsimony method, and one distance matrix tree, using the neighbour joining method. Petunia sensu Wijsman and Calibrachoa were monophyletic sister clades in both trees. Calibrachoa parviflora and C. pygmaea, previously thought to differ from the other species in terms of their cross-compatibility, seed morphology, and nuclear DNA content, formed a basal clade that was sister to the remainder of Calibrachoa. Several clades found in the phylogenetic trees corresponded to their distribution ranges, suggesting that recent speciation in the genus Petunia sensu Jussieu occurred independently in several different regions. CONCLUSIONS: The separation of Petunia sensu Wijsman and Calibrachoa was supported by chloroplast DNA analysis. Two groups in the Calibrachoa were also recognized with a high degree of confidence.     

Phylogenetic analysis of the non-pathogenic genusSpiromastix (Onygenaceae) and related onygenalean taxa based on large subunit ribosomal DNA sequences

The phylogenetic positioning of the non-pathogenic genusSpiromastix in the Onygenales was studied based on large subunit rDNA (LSU rDNA) partial sequences (ca. 570 bp.). FourSpiromastix species and 28 representative taxa of the Onygenales were newly sequenced. Phylogenetic trees were constructed by the neighbor-joining (NJ) method and evaluated by the maximum parsimony (MP) method with the data of 13 taxa retrieved from DNA databases.Spiromastix and dimorphic systemic pathogens,Ajellomyces andParacoccidioides, appear to be a monophyletic group with 74% bootstrap probability (BP) in the NJ tree constructed with the representative taxa of the Onygenales. The tree topology was concordant with the NJ tree based on SSU rDNA sequences of our previous work and corresponded to the classification system of the Onygenales by Currah (1985) and its minor modification by Udagawa (1997) with the exception of the classification of the Onygenaceae. The Onygeneceae sensu Udagawa may still be polyphyletic, since three independent lineages were recognized. The taxa forming helicoid peridial appendages were localized to two clades on the tree. The topology of the NJ tree constructed withSpiromastix and its close relatives suggested that the helicoid peridial appendages were apomorphic and acquired independently in the two clades of the Onygenales.     

Phylogeny of the superfamily Gelechioidea (Lepidoptera: Obtectomera), with an exploratory application on geometric morphometrics

The superfamily Gelechioidea (Lepidoptera: Obtectomera) has a high species diversity. It consists of more than 18,400 described species and has a global distribution. Among it, large numbers of species were reported to be economically important to people's production and life. However, relationships among families or subfamilies in Gelechioidea have been exceptionally difficult to resolve using morphology or single gene genealogies. Multiple gene genealogies had been used in the molecular phylogenetic studies on Gelechioidea during the past years, but their phylogenetic relationships remain to be controversial mainly due to their limited taxa sampling relative to such high species diversity. In this paper, 89 ingroup species representing 55 genera are sequenced and added to the data downloaded from GenBank, and six species representing four closely related superfamilies are chosen as outgroup. The molecular phylogeny of Gelechioidea is reconstructed based on the concatenated data set composed of one mitochondrial marker (COI) and seven nuclear markers (CAD, EF-1ɑ, GAPDH, IDH, MDH, RpS5, wingless). The phylogenetic results, taking into consideration of the comparative morphological study, show that the clade of Gelechioidea is strongly supported and separated from other superfamilies, which further proves its monophyly. Five families are newly defined: Autostichidae sensu nov., Depressariidae sensu nov., Peleopodidae sensu nov., Ashinagidae sensu nov. and Epimarptidae sensu nov. Meanwhile, a monophyletic “SSABM” clade considered to be closely related is proposed for the first time, consisting of Stathmopodidae, Scythrididae, Ashinagidae, Blastobasidae and Momphidae. Moreover, geometric morphometric analyses using merged landmark data set from fore and hind wings of 118 representative species are conducted. The phenetic tree shows that the monophyly and phylogenetic relationships correspond with the results of molecular phylogeny largely, which well proves its importance and potential application in both phylogenetic reconstruction and species identification.     

Genetic structuring of important medicinal species of genus Warburgia as revealed by AFLP analysis

The genus Warburgia (Canellaceae) contains four tree species that are of valuable medicinal importance and are all found in Africa. Genetic diversity present in wild populations of these species is under great threat due to unsustainable harvesting for medicines and indiscriminate felling for timber and agricultural expansion. There is therefore an urgent need for conservation of these species. Some authors disagree about the taxonomy of the genus and list different species as synonyms. Amplified fragment length polymorphism (AFLP) technique was used to determine the genetic relationships between three species to resolve the taxonomic confusion. The amount of genetic variation within and among populations was assessed to guide strategies for their conservation and sustainable utilization. Four AFLP primer pairs (EcoRI/MseI) generated a total of 185 amplification products. Analysis of molecular variance revealed most variation among individuals within populations (63%, P < 0.0001), but variation among populations (37%, P < 0.0001) was highly significant as well. Constrained analysis of principal coordinates based on the Jaccard distance confirmed the separation among populations (38.2%, P < 0.0001). A phenetic tree and ordination graphs showed a clear distinction of W. ugandensis from W. salutaris and W. stuhlmannii. W. ugandensis populations from Uganda and western Kenya formed a subgroup that clustered away from the rest of the W. ugandensis populations. W. salutaris and W. stuhlmannii populations showed little genetic differentiation. An implication of the data to genetic management and taxonomic clarification is discussed.     

Systematics of Maxomys Sody, 1936. (Rodentia: Muridae: Murinae): DNA/DNA hybridization studies of some Borneo-Javan species and allied Sundaic and Australo-Papuan genera

We compared five species of the murine genus Maxomys and representatives of nine other murid genera in a complete 15 × 15 DNA-hybridization matrix. FITCH trees were calculated for the entire suite of taxa and for subsets including only the five Maxomys and these together with the four nearest outgroups. All trees were validated by 'bootstrapping' and by jackknifing, performing both single- and multiple-deletions of taxa. The full 15 times 15 data set indicated a sister-group relationship between Maxomys and two pairs of genera ( Sundamys-Rattus sensu stricto and Mviventer-Leopoldamys ) that are more closely related to each other than to Maxomys; addition of data on Bandicota and Berylmys from another recent DNA-hybridization study confirmed that these genera are successive sister-taxa to the Sundamys-Rattus pair. Mus-Myomys and Uromys-Melomys were each distinct lineages from the above grouping of Rattus sensu lata species, and from the putative outgroup sigmodontine Peromyscus, but the interrelations of the three murine clades were unresolved. Within Maxomys, M. surifer and M. bartelsii are a related pair, and M. ochraceiventer probably forms an unresolved trio with M. rajah and M. whiteheadi. Calibration of a tree generated from saturation-corrected distances against a likely divergence-date of 12.2 Mybp for the separation of Mus and Rattus confirms a high rate of single-copy DNA change in murids (2.1%/Myr); and suggests that Sigmodontinae and Murinae diverged at around 15.3 Mybp, that Maxomys and the group of six other Rattus sensu lato separated approximately 7.6 Myr ago, and that Maxomys began to diversify 4.8 Myr ago.     

Phylogenetic relationships among Secale species revealed by amplified fragment length polymorphisms.

Amplified fragment length polymorphism (AFLP) data were utilized to analyze the phylogenetic relationships among 29 accessions representing 14 of the most commonly recognized ranked species or subspecies in the genus Secale. We observed 789 AFLP markers of 1130 fragments utilizing 18 P-/M- and E-/M- primer combinations. All polymorphic fragments were used to construct phenetic and phylogenetic trees. The resulting phenogram and cladogram had similar tree topologies. Cluster analysis showed that Secale sylvestre was the most distantly related to all other ryes. Annual forms were grouped together, and the perennial forms appeared more closely related to each other. This suggested that life cycle could have played an important role in determining the relationships among Secale species. Secale sylvestre was considered to be the most ancient species, whereas Secale cereale was the most recently evolved species. Amplified fragment length polymorphism analysis clearly separated all Secale species into only 3 major species groups, within the genus Secale: S. sylvestre, Secale montanum (syn. Secale strictum) for perennial forms, and S. cereale for annual forms. This study demonstrated that the AFLP approach is a useful tool for discriminating species differences, and also gave a much better resolution in discerning genetic relationships among Secale species as compared with previous studies using other approaches.     

A PHYLOGENETIC STUDY OF THE CORALLINALES (RHODOPHYTA) BASED ON NUCLEAR SMALL-SUBUNIT rRNA GENE SEQUENCES

Conflicting classifications for the Corallinales were tested by analyzing partial sequences for the nuclear small-subunit ribosomal RNA (SSU) gene of 35 species of coralline algae. Parsimony and likelihood analyses of these data yielded congruent hypotheses that are inconsistent with classifications for the group that include as many as eight subfamilies. Four major clades are resolved within the order, including the early-diverging Sporolithaceae as well as the Melobesioideae and Corallinoideae. The fourth clade, which is supported robustly, includes both nongeniculate and geniculate species classified in the subfamilies Mastophoroideae, Metagoniolithoideae, Lithophylloideae, and Amphiroideae. Molecular and morphological data support the proposal that the latter two subfamilies are sister taxa. Although relationships among some genera are not resolved clearly, the order of branching of taxa among and within the four principal lineages is concordant with paleontological evidence for the group. Relationships inferred among genera within each of the clades is discussed. Seven morphological characters delimiting higher taxonomic groups within the order were combined with the sequence data, analyzed, and optimized onto the resulting tree(s). Except for the presence or absence of genicula, all other characters were found to be phylogenetically informative. Genicula are nonhomologous structures that evolved independently in the Amphiroideae, Corallinoideae, and Metagoniolithoideae. The phenetic practice of separating coralline algae into two categories solely on the basis of the presence or absence of genicula does not accurately reflect the evolutionary history of the group.     

Snake relationships revealed by slow-evolving proteins: a preliminary survey

We present an initial evaluation of relationships among a diverse sample of 215 species of snakes (8% of the world snake fauna) representing nine of the 16 commonly-recognized families. Allelic variation at four slow-evolving. protein-coding loci, detected by starch-gel electrophoresis, was found to be informative for estimating relationships among these species at several levels. The numerous alleles detected at these loci [ Arp -2 (42 alleles). Ltlh -2 (43), Mdh -1 (29), Pgm (Z)] provided unexpected clarity in partitioning these taxa. Most congeneric species and several closely-related genera have the same allele at all four loci or differ at only a single locus. At thc other extreme are those species with three or four unique alleles; these taxa cannot be placed in this analysis. Species sharing two or three distinctive alleles are those most clearly separated into clades. Typhlopids, pythonids, viperids, and elapids were resolved into individual clades. whereas bods were separated into boincs and erycines, and colubrids appeared as scveral distinct clades (colubrines, natricines, psammophines, homalopsines, and xenodontines). Viperids were recognized as a major division containing three separate clades: Asian and American crotalines. Pabearctic and Oriental viperines, and Ethiopian causines. The typhlopids were found to be the basal clade, with the North American erycine boid Chrrrino and the West Indian woodsnakes Tropidophi, Y near the base. A number of species and some small clades were not allocated because of uninformative (common, unique, or conflicting) alleles. Of the 21 S species examined, five to eight appear to have been misplaced in the analysis of these electrophoretic data.