Genetic diversity, structure, and patterns of differentiation in the genus Vitis

Vitis (Vitaceae) is a taxonomically complicated genus with ca. 60 taxa divided into two subgenera, Vitis and Muscadinia. We used population genetic approaches to gain insights into the genetic diversity, patterns of evolutionary differentiation and to decipher the taxonomic status of some of the controversial taxa within the genus Vitis. The distance- and model-based analyses were used to examine the phylogenetic structure within the genus Vitis using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. The results closely matched the current classification, but some discrepancies in the identity of taxa at the specific and subspecific levels were still evident. The East Asia and the North American Vitis exhibited strong divergence and each group showed further differentiation into several subgroups with North American subgroups roughly matching the described series. The model based cluster analysis indicated 14 clusters as optimum to explain the genetic structure within the genus Vitis with most clusters containing a moderate frequency of admixed genotypes suggesting interspecific gene flow within the subgenus Vitis. Hierarchical partitioning of molecular variation indicated that a significant amount of the total variation (~74 % and ~69 % for SSRs and AFLPs, respectively) is accounted for by intraspecific variation as compared to the levels due to genetic differentiation among species within series (~17 % and ~20 % for SSRs and AFLPs, respectively) and among series within the genus Vitis (~9 % and ~10 % for SSRs and AFLPs, respectively). Overall, Vitis possesses mild genetic structure characterized by reticulation and incomplete lineage sorting of ancestral polymorphisms.     

Evolutionary history of almond tree domestication in the Mediterranean basin

Genetic diversity of contemporary domesticated species is shaped by both natural and human‐driven processes. However, until now, little is known about how domestication has imprinted the variation of fruit tree species. In this study, we reconstruct the recent evolutionary history of the domesticated almond tree, Prunus dulcis, around the Mediterranean basin, using a combination of nuclear and chloroplast microsatellites [i.e. simple sequence repeat (SSRs)] to investigate patterns of genetic diversity. Whereas conservative chloroplast SSRs show a widespread haplotype and rare locally distributed variants, nuclear SSRs show a pattern of isolation by distance with clines of diversity from the East to the West of the Mediterranean basin, while Bayesian genetic clustering reveals a substantial longitudinal genetic structure. Both kinds of markers thus support a single domestication event, in the eastern side of the Mediterranean basin. In addition, model‐based estimation of the timing of genetic divergence among those clusters is estimated sometime during the Holocene, a result that is compatible with human‐mediated dispersal of almond tree out of its centre of origin. Still, the detection of region‐specific alleles suggests that gene flow from relictual wild preglacial populations (in North Africa) or from wild counterparts (in the Near East) could account for a fraction of the diversity observed.     

An assessment of the genetic diversity of Cedrela balansae C. DC. (Meliaceae) in Northwestern Argentina by means of combined use of SSR and AFLP molecular markers

Cedrela balansae C.DC. is a native tree species in Argentina, severely exploited for its timber features. We performed a molecular analysis to understand the genetic diversity and its distribution in eight remaining populations, which are distributed within the species' range in the Argentine Yungas Rainforest. We used two molecular markers: (i) seven SSRs, selected from forty-five SSRs developed for phylogenetically close species belonging to the Meliaceae family and (ii) 382 polymorphic AFLPs. The He was 0.643 and 0.222 for SSRs and AFLPs, respectively. The moderate levels of genetic diversity were related to the limited size of the species' distribution area, the latitudinal position of populations, the impacts of logging and the species' spatial distribution pattern. Genetic differentiation among populations was low for both markers (4.9% and 4.1% for SSRs and AFLPs, respectively). Four genetic clusters homogeneously distributed were distinguished. These observations may relate to the considerable historical gene flow measured (3.71 and 4.47 for SSRs and AFLPs, respectively). To safeguard the currently existing genetic base in the species, we identify four priority populations for conservation. To date only one of these is located in a protected area. Therefore, it is urgent to apply additional conservation measures for the remaining populations.     

Pros and cons of using genomic SSRs and EST-SSRs for resolving phylogeny of the genus Gossypium

The genus Gossypium is comprised of 50 diverse cotton species representing eight different genomes (A through G and K), however, phylogenetic relationship using various DNA marker types such as RAPD and SSRs was determined on limited number of cotton species. In this report, we have demonstrated the application of genomic SSRs (gSSRs) and EST-SSRs, and after combining both the data sets, for resolving the phylogenies of 36 cotton species including seven races. Out of the 100 primer pairs surveyed (50 for gSSRs and 50 for EST-SSRs), 75 produced scorable amplification products in all species. Out of these, 73 were found to be polymorphic and amplified 135 alleles ranging from 1 to 5 alleles per SSR marker (average 2.87 alleles per marker). The gSSRs amplified higher number of alleles (72) compared to the EST-SSRs (63). In total 22 highly informative SSRs with PIC values ≥0.5 were identified. Genomic SSRs containing di-while EST-SSRs containing tri-nucleotide repeats exhibited high polymorphism compared to the other nucleotide repeats containing gSSRs/EST-SSRs. Number of tandem repeats and polymorphism were positively correlated. Neither the type of chromosome nor the location of the SSRs showed association with the polymorphism. Gossypium herbaceum var. africanum (Watt) Hutch. ex and Ghose and Gossypium robinsonii F. Muell. were found the most genetically diverse, while among races of Gossypium hirsutum L. “yucatanense” and G. hirsutum “punctatum” were found genetically diverse. Of the three data sets, clustering analysis based on EST-SSRs and combined data sets, revealed parallel results reported in earlier studies. This study further confirmed that Gossypium darwinii Watt has close relationship with Gossypium barbadense L. Moreover, Gossypium raimondii Ulbr. and G. herbaceum/Gossypium arboreum L. are close living relatives of the ancestor allotetraploid species. Our studies suggest that for resolving phylogenetic relationship among the various plant species EST-SSRs could be a better choice. This information can be instrumental in transferring novel alleles or loci from the wild species into the cultivated cotton species which would set a stage for cultivating genetically diverse cultivars—a way to achieve sustainable cotton production in changing climate.     

Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter- to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine the taxonomy of this group, which may illuminate why some barcoding criteria fail to reliably differentiate species.     

Phylogeny of woodcreepers of the genus Lepidocolaptes (Aves,Furnariidae), a widespread Neotropical taxon

Arbeláez‐Cortés, E., Navarro‐Sigüenza, A. G. & García‐Moreno J. (2012). Phylogeny of woodcreepers of the genus Lepidocolaptes (Aves, Furnariidae), a widespread Neotropical taxon. —Zoologica Scripta, 41, 363–373. Phylogeny of woodcreepers of the genus Lepidocolaptes (Aves, Furnariidae), a widespread Neotropical taxon. The phylogeny of the genus Lepidocolaptes was reconstructed based on three mitochondrial DNA regions and one nuclear DNA intron, using Bayesian analysis. A general pattern of diversification among the lowland species followed by the diversification of highland species, and a close relationship among montane species with the two Atlantic Forest endemics, seem to depict the history of this genus. Results also showed that the two Mesoamerican species are sister‐taxa with high support. Finally, our data also suggest the existence of previously unknown intraspecific genetic structure within some taxa, especially among populations of Lepidocolaptes souleyetii.     

Functional Genetic Diversity Analysis and Identification of Associated Simple Sequence Repeats and Amplified Fragment Length Polymorphism Markers to Drought Tolerance in Lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> ssp. <Emphasis Type="Italic">culinaris</Emphasis> Medicus) Landraces

Genetic diversity of 70 Mediterranean lentil (Lens culinaris ssp. culinaris Medicus) landraces was assessed using simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs). These landraces were also assessed for variation in root and shoot traits and drought tolerance as estimated by relative water content (RWC), water losing rate (WLR) and wilting score (WS). Genetic diversity and clear differentiation of Moroccan landraces from those from northern Mediterranean regions (Italy, Turkey and Greece) were found. High genetic variation in root and shoot traits and traits related to drought tolerance was also observed. No relationship was found between drought tolerance of landraces and their geographic origin. Landraces with higher dry root biomass, chlorophyll content and root–shoot ratio were drought tolerant as evidenced by higher RWC and lower WLR and wilting severity. Kruskal–Wallis non-parametric test (K-W) was used to find SSRs and AFLPs associated with RWC, WLR and WS. Regression analysis showed six SSR and AFLP alleles explaining the highest phenotypic variation of RWC, WLR and WS (ranging from 21 to 50 % for SSRs and from 14 to 33 % for AFLPs). Functional genetic diversity analysis showed relationships between drought response of landraces and linked SSR and AFLP alleles to RWC, WLR and WS according to K-W test using canonical discriminant analysis. Our results confirm the feasibility of using association mapping to find DNA markers associated with drought tolerance in larger numbers of lentil landraces.     

Development and Application of Microsatellites in Candidate Genes Related to Wood Properties in the Chinese White Poplar (Populus tomentosa Carr.)

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2–7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.     

Phylogenetic significance of the characteristics of simple sequence repeats at the genus level based on the complete chloroplast genome sequences of Cyatheaceae

The simple sequence repeats (SSRs) of plant chloroplasts show considerable genetic variation and have been widely used in species identification and phylogenetic relationship determination. Whether chloroplast genome SSRs can be used to classify Cyatheaceae species has not yet been studied. Therefore, the chloroplast genomes of eight Cyatheaceae species were sequenced, and their SSR characteristics were compared and statistically analyzed. The results showed that the chloroplast genome structure was highly conserved (genome size: 154,046–166,151 bp), and the gene content (117 genes) and gene order were highly consistent. The distribution characteristics of SSRs (number, relative abundance, relative density, GC content) showed taxon specificity. The primary results were the total numbers of SSRs and mononucleotides: Gymnosphaera (61–67 and 40–47, respectively), Alsophila (121–122 and 95–96), and Sphaeropteris (102–103 and 77–80). Statistical and clustering analyses of SSR characteristics showed that their distribution was consistent with the recent classification of Cyatheaceae, which divided the eight Cyatheaceae species into three genera. This study indicates that the distribution characteristics of Cyatheaceae chloroplast SSRs can provide useful phylogenic information at the genus level.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Identification and Phylogenetic Classification of <Emphasis Type="Italic">Pennisetum</Emphasis> (Poaceae) Ornamental Grasses Based on SSR Locus Polymorphisms

Pennisetum species are widely used as ornamental grasses and may be a valuable genetic resource for their breeding to broaden its genetic basis. At present, new ornamental Pennisetum cultivars are primarily bred via somaclone, which increases the number of variants. It is difficult to estimate whether the suspected variants are authentic in genetic features by morphological traits because of their many limitations. Moreover, although the phylogenetic classification of the Pennisetum genus has been approved in some morphological and cytological studies, genetic evidence is lacking. In the present study, we developed 15 specific simple sequence repeat (SSR) markers with a large amount of polymorphisms and strong distinguishing abilities for Pennisetum ornamental grasses using magnetic bead enrichment. These markers, together with the other 11 reported polymorphic SSRs, were further used for the identification of a broad collection of 55 Pennisetum samples, including nine original taxa and 46 suspected variants. After comparing the genetic characteristics between each variant and its corresponding original taxon, we verified 20 suspected variants that possess the potential to become new, commercially desirable cultivars. The nine original taxa and the 20 verified variants were identified based on the polymorphisms of six core loci, and unique molecular identities with 15 denary digits for each taxon were further established. The rationality of the traditional phylogenetic classification system of the Pennisetum genus was further verified using 147 polymorphic alleles. The present study promotes the protection, registration, breeding, and international communication of Pennisetum ornamental grasses.     

Transferability of olive microsatellite loci across the genus <Emphasis Type="Italic">Olea</Emphasis>

The transferability of microsatellite markers developed for olive cultivars (Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.Communicated by H.F. Linskens     

Molecular variation and population structure in endangered <Emphasis Type="BoldItalic">Limonium</Emphasis><Emphasis Type="BoldItalic">bicolor</Emphasis>: genetic diversity of microsatellite markers and amplified fragment length polymorphism analysis

Knowledge and analysis of the genetic structure of an endangered species is important for its conservation and evolutionary process. Simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs) were used in evaluation of the genetic diversity and population differentiation in Limonium bicolor (Plumbaginaceae), an endangered herb with high medicinal and horticulture value. A total of 117 alleles were detected with an average 5.85 alleles per locus using SSR and 222 bands from AFLP were amplified in six populations. It was found that L. bicolor was characterized by high levels of genetic polymorphism (100 and 83.78%), low levels of total genetic diversity (\(H_{\mathrm{t}}=\) 0.2824 and 0.2424), and moderate degrees of genetic differentiation among populations (\(\Phi _{\mathrm{ST}} =\) 0.284 and 0.251). Analysis of molecular variance (AMOVA) revealed that the main variation component existed within populations (71.56%; 74.93%) rather than among populations (28.44%; 25.07%). Four main clusters were displayed in the UPGMA using TFPGA, which was consistent with the result of principal coordinate analysis (PCA) using NTSYS. Mutations or infrequent gene flow among populations can increase the plant slowly, thus in situ conservation policies should be implemented first for effective and sustainable development. At the same time, ex situ measures, such as those individuals with rare alleles, to maintain the relationships between individuals and populations are also proposed.     

POPULATION GENETICS AND TAXONOMY OF CAULERPA (CHLOROPHYTA) FROM THE GREAT BARRIER REEF,AUSTRALIA1

Allozyme variation was examined in seven species and four varieties of Caulerpa sampled from the Great Barrier Reef (GBR) region, Australia. Differences between species were greater than those between populations of the same taxon sampled from different geographical locations, and typically included fixed gene differences (no alleles found in one taxon shared by the other taxon) at two or more loci. Three varieties of C. racemosa, vars imbricata, laetevirens, and racemosa, and a peltate morph of C. racemosa were as strongly distinguished genetically as the six other species examined: C. cupressoides, C. lentillifera, C. peltata, C. serrulata, C. sertularioides, and C. taxifolia. Cluster analysis did not place all of the C. racemosa varieties together and linked them with other species, but cladistic analyses showed the allozyme data gave little useful phylogenetic information. Eight of 13 plants identified initially as C. serrulata were distinguished at several loci, indicating the presence of an undetermined cryptic taxon. Population genetic analysis of polymorphism, which occurred in some taxa, demonstrated strong spatial differentiation among populations of C. cupressoides, C. racemosa vars laetevirens and racemosa, C. serrulata, and C. taxifolia and significant but variable degrees of clonality and/or inbreeding within these populations. Allozymes proved to be a useful tool for defining species boundaries and investigating population structure in Caulerpa, but not for determining phylogenetic relationships within the genus.     

Genetic diversity and population structure of Buddleja crispa Bentham in the Himalaya-Hengduan Mountains region revealed by AFLP

Buddleja crispa Benth. is one of the most morphologically variable species in genus Buddleja, and it is widely distributed in the Himalaya-Hengduan Mountains (HHM) region. This study used AFLPs as a tool to examine the genetic variation among and within 25 populations of B. crispa. Analysis of population genetics of the species aimed to clarify morphological variation, current distribution patterns, strong adaptability to habitats, and the effects of geological factors in the HHM region. The genetic structure results, based on PCoA and NJ cluster analyses, revealed that the populations of B. crispa were divided into two genetic groups. Furthermore, the peripheral populations had lower genetic diversity than the populations in the center of the distribution areas (Three Parallel Rivers). We conclude that the gene flow (predominantly seed and pollen flow) and the population differentiation of B. crispa might be more affected by the barriers formed by rivers and mountains than by geographic distance.     

Population Genetics of the Rubber-Producing Russian Dandelion (Taraxacum kok-saghyz)

The Russian dandelion, Taraxacum kok-saghyz (TKS), is a perennial species native to Central Asia that produces high quality, natural rubber. Despite its potential to help maintain a stable worldwide rubber supply, little is known about genetic variation in this species. To facilitate future germplasm improvement efforts, we developed simple-sequence repeat (SSR) markers from available expressed-sequence tag (EST) data and used them to investigate patterns of population genetic diversity in this nascent crop species. We identified numerous SSRs (1,510 total) in 1,248 unigenes from a larger set of 6,960 unigenes (derived from 16,441 ESTs) and designed PCR primers targeting 767 of these loci. Screening of a subset of 192 of these primer pairs resulted in the identification of 48 pairs that appeared to produce single-locus polymorphisms. We then used the most reliable 17 of these primer pairs to genotype 176 individuals from 17 natural TKS populations. We observed an average of 4.8 alleles per locus with population-level expected heterozygosities ranging from 0.28 to 0.50. An average pairwise F_ST of 0.11 indicated moderate but statistically significant levels of genetic differentiation, though there was no clear geographic patterning to this differentiation. We also tested these 17 primer pairs in the widespread common dandelion, T. officinale, and a majority successfully produced apparently single-locus amplicons. This result demonstrates the potential utility of these markers for genetic analyses in other species in the genus.     

Phytosociological affiliation of Annex II species Tephroseris longifolia subsp. moravica in comparison with two related Tephroseris species with overlapping distribution

The phytosociological affiliation of Tephroseris longifolia subsp. moravica, species of European importance, was studied in relation to two closely related species of the genus Tephroseris which have overlapping distribution within the Western Carpathian Mts: T. intergrifolia and T. crispa. The main aim was to compare plant communities inhabited by the three taxa, to assess the major environmental gradients responsible for variation in their distribution and to estimate ecological indicator values for Tephroseris longifolia subsp. moravica. T. longifolia subsp. moravica was recorded in nine localities in the Slovakia and Czech Republic where it occurs in very specific site conditions of ecotone habitats. Its phytosociological affiliation is restricted to grasslands of the alliances Bromion erecti and Arrhenatherion elatioris and to the ecotone vegetation between these grasslands and beech forests. T. integrifolia occurs most frequently in the Diantho lumnitzeri-Seslerion, Bromion erecti and Quercion pubescenti-petraeae alliances. T. crispa occurs predominantly in communities of the Calthion palustris alliance and Scheuchzerio-Caricetea fuscae, Mulgedio-Aconitetea and Montio-Cardaminetea classes. The major gradient responsible for variation in species composition of communities inhabited by the studied taxa was associated with moisture and nutrient content. The vascular plant-based ecological indicator values for Tephroseris longifolia subsp. moravica calculated from phytosociological relevés with its occurrence were set for light — 6, temperature — 5, continentality — 4, moisture — 5, soil reaction — 6 and nutrients — 5. We conclude that the studied taxon has intermediate relationship to the most of the studied factors in comparison with two related species, T. crispa and T. integrifolia.     

Relationships among the major lineages of Dictyoptera: the effect of outgroup selection on dictyopteran tree topology

Abstract Dictyoptera, comprising Blattaria, Isoptera, and Mantodea, are diverse in appearance and life history, and are strongly supported as monophyletic. We downloaded COII, 16S, 18S, and 28S sequences of 39 dictyopteran species from GenBank. Ribosomal RNA sequences were aligned manually with reference to secondary structure. We included morphological data (maximum of 175 characters) for 12 of these taxa and for an additional 15 dictyopteran taxa (for which we had only morphological data). We had two datasets, a 59‐taxon dataset with five outgroup taxa, from Phasmatodea (2 taxa), Mantophasmatodea (1 taxon), Embioptera (1 taxon), and Grylloblattodea (1 taxon), and a 62‐taxon dataset with three additional outgroup taxa from Plecoptera (1 taxon), Dermaptera (1 taxon) and Orthoptera (1 taxon). We analysed the combined molecular?morphological dataset using the doublet and MK models in Mr Bayes , and using a parsimony heuristic search in paup . Within the monophyletic Mantodea, Mantoida is recovered as sister to the rest of Mantodea, followed by Chaeteessa; the monophyly of most of the more derived families as defined currently is not supported. We recovered novel phylogenetic hypotheses about the taxa within Blattodea (following Hennig, containing Isoptera). Unique to our study, one Bayesian analysis places Polyphagoidea as sister to all other Dictyoptera; other analyses and/or the addition of certain orthopteran sequences, however, place Polyphagoidea more deeply within Dictyoptera. Isoptera falls within the cockroaches, sister to the genus Cryptocercus. Separate parsimony analyses of independent gene fragments suggest that gene selection is an important factor in tree reconstruction. When we varied the ingroup taxa and/or outgroup taxa, the internal dictyopteran relationships differed in the position of several taxa of interest, including Cryptocercus, Polyphaga, Periplaneta and Supella. This provides further evidence that the choice of both outgroup and ingroup taxa greatly affects tree topology.     

Genetic Differentiation Among Annual Fishes of the Genus Cynolebias (Cyprinodontiformes, Rivuluidae) in a Biosphere Reserve Site from Uruguay

The annual killifishes of the genus Cynolebias include one well supported clade composed of three taxa: Cynolebias adloffi, from Porto Alegre (southern Brazil), C. cf. adloffi and Cynolebias viarius, parapatrically distributed in southeastern Uruguay. Cynolebias cf. adloffi has always been considered an intergradation complex of populations between C. adloffi and C. viarius showing high levels of morphological variability and its systematic affinities have been historically discussed. This study clarified the patterns of genetic differentiation of C. cf. adloffi by means of RAPD markers to define its taxonomic status as a possible separated taxon. Multidimensional scaling analysis reveals that C. cf. adloffi is composed of two highly structured genetic groups (northern and southern) with discrete geographical distributions. The estimate of gene diversity for RAPD markers could suggest a scenario in which the rainy season flood pool populations across large distances could make intrapopulation variation high but interpopulation variation low. Phylogenetic analyses (UPGMA and parsimony) show different and unstable patterns of relationships among the taxa analyzed. In the UPGMA tree, C. viarius and C. cf. adloffi appear as sister taxa, while parsimony analysis does not support a unique hypothesis of relationship. Our analyses are in agreement with the hypothesis that C. cf. adloffi may well be a case of ancient hybrid ancestry followed by its divergence associated with Pleistocene and post-Pleistocene environmental changes in this region.