Evolution of the Gibbon Subgenera Inferred from CytochromebDNA Sequence Data

DNA sequences for the mitochondrial cytochromebgene from the four extant gibbon subgenera are described. The data confirm that the gibbon subgenera evolved from a common hylobatid ancestor and suggest that they diverged from each other after the divergence of the extant African great ape species. The cytochromebgene does not resolve the evolutionary relationships between the gibbon subgenera themselves.     

Phylogenetic relationships among the skunks: A molecular perspective

Phylogenetic relationships among the three recognized genera of skunks (Mephitis, Spilogale, andConepatus) were examined using allozymes and nucleotide sequence data from portions of the cytochromeb gene and displacement loop of the mitochondrial genome. The data sets indicated thatMephitis andSpilogale shared a common ancestor after they diverged fromConepatus. Intrageneric relationships revealed that the level of genetic divergence between the two species ofSpilogale was equal to the level of divergence between the two species ofMephitis, with the two North American species ofConepatus possessing the least amount of genetic divergence.     

Intrageneric Phylogeny ofAcomys(Rodentia, Muridae) Using Mitochondrial Gene Cytochromeb

This paper investigates interspecies relationships within the genusAcomys(spiny mice) by analyzing entire mitochondrial cytochromebgene (1141 bp). This gene provides strong phylogenetic signal, as shown by high support of the topology obtained (bootstrap value and RNA support number). The phylogeny is congruent with inferences from allozymes for the species considered. Controversial taxonomy ofAcomys cahirinus, dimidiatus, airensis,andignitusis clarified, with their specific ranks confirmed on the basis of tree topology and nucleotide distances. Phylogenetic relationship between the undescribed speciesAcomyssp. from west Africa andA. airensisargue in favor of two distinct colonization events in this zone.     

Genista tinctoria microsymbionts from Poland are new members of Bradyrhizobium japonicum bv. genistearum

The phylogeny and taxonomic position of slow-growing Genista tinctoria rhizobia from Poland, Ukraine and England were estimated by comparative 16S rDNA, atpD, and dnaK sequence analyses, PCR-RFLP of 16S rDNA, DNA G + C content, and DNA–DNA hybridization. Each core gene studied placed the G. tinctoria rhizobia in the genus Bradyrhizobium cluster with unequivocal bootstrap support. G. tinctoria symbionts and bradyrhizobial strains shared 96–99% similarity in 16S rDNA sequences. Their similarity for atpD and dnaK sequences was 93–99% and 89–99%, respectively. These data clearly showed that G. tinctoria rhizobia belonged to the genus Bradyrhizobium. 16S rDNA sequence analysis was in good agreement with the results of the PCR-RFLP of the 16S rRNA gene. Although the tested strains formed separate lineages to the reference bradyrhizobia their RFLP 16S rDNA patterns were quite similar. The genomic DNA G + C content of three G. tinctoria rhizobia was in the range from 60.64 to 62.83 mol%. Data for species identification were obtained from DNA–DNA hybridization experiments. G. tinctoria microsymbionts from Poland were classified within Bradyrhizobium japonicum genomospecies based on 56–82% DNA–DNA similarity.     

Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.     

Morphometric, biochemical and molecular traits in Caucasian wood mice (podemus/Sylvaemus), with remarks on species divergence

We analysed Caucasian wood mice from Georgia (n = 60) and supplementary reference material of theApodemus/Sylvaemus species group to evaluate the reliability of taxon identification. Traditional “expert knowledge” plus three different methodological approaches were employed and combined to perceive their discriminatory power for a reliable taxon assignment. Graphs of principal component scores derived from the analysis of 14 skull metrics displayed taxon membership of individuals. Individual multi--locus (L = 18) electrophoretic profiles were used to re-assess specimens to a specific genepool by an assignment test based on allele frequencies indicative of populational taxon samples of the respective sampling locations. Genotyped individuals were re-allocated to those taxa, for which they yielded the highest probability score. Genetic distances among the taxa were computed and clustered in a neighbour-joining tree. PCR-fragments of 1074bp amplified from the mitochondrial cytochromeb gene were cut with 2 six- and 4 four-cutter restriction enzymes, and resulting RFLP patterns were analysed phenetically to classify the specimens according to their molecular similarity. Partial cytochromeb sequences were used to construct a phylogenetic tree by computing neighbour-joining clusters from a matrix of percent nucleotide differences. The power of the combined classification approaches and their congruence is discussed. It is concluded that the joint application of traditional, morphometric and biochemical or genetic techniques for taxon allocation of specimens of wood mice encountered problems in species delimitation. The mtDNA topology obtained was not congruent with protein polymorphism that indicated differential historical and/or recent introgression and incomplete lineage sorting in substructured populations. Cytochromeb sequence DNA data analysed were not as adequate as expected to resolve phylogenetic relationships among Caucasian and European members of theApodemus-Sylvaemus complex. Altogether, morphometric, biochemical and sequence data sets did not support the hypothesis of the evolutionary independence of European and Caucasian lineages of wood mice. Nonetheless, extended combined morphological and genetic analyses are considered necessary prerequisites to an in-depth study of the evolutionary lineages of theApodemus/Sylvaemus group. More sequence data of a variety of genes (and plenty of nuclear markers) are needed to resolve the various levels of differentiation of the extant lineages.     

Finding Optimal Ingroup Topologies and Convexities When the Choice of Outgroups Is Not Obvious

Considerable confusion remains among theoreticians and practicioners of phylogenetic science on the use of outgroup taxa. Here, we show that, despite claims to the contrary, details of the optimal ingroup topology can be changed by switching outgroup taxa. This has serious implications for phylogenetic accuracy. We delineate between the process of outgroup selection and the various possible processes involved in using an outgroup taxon after one has been selected. Criteria are needed for the determination that particular outgroup taxa do not reduce the accuracy of evolutionary tree topologies and inferred character state transformations. We compare previous results from a sensitivity bootstrap analysis of the mitochondrial cytochromebphylogenetic relationships among whales to the results of a Bremer support sensitivity analysis and of a recently developed application of RASA theory to the question of putative outgroup taxon plesiomorphy content.     

Phylogenetic relationships of Hamadryas (Nymphalidae: Biblidinae) based on the combined analysis of morphological and molecular data

A new phylogenetic hypothesis for the Neotropical butterfly genus Hamadryas, based on the combination of a morphological matrix, one mitochondrial (COI) and four nuclear markers (CAD, RpS5, EF1a, and Wingless), is presented. Results from analyses of the molecular evidence are compared with a previously published morphological phylogeny. Molecular data and the analysis of the complete dataset support the monophyly of Hamadryas and most sister groups suggested by morphological data alone. The addition of DNA sequences to the morphological matrix helped define species groups for which no morphological synapomorphies were found. Partitioned Bremer support indicates that COI, CAD, and morphology were consistently in agreement with the combined evidence tree. In contrast, signal from the nuclear markers Rps5, EF1a, and Wingless showed indifference at most levels of the tree, and minor conflict at nodes solving the relationships between species groups. Though resolved, the combined evidence tree shows low resample values, particularly among species groups whose relationships were characterized by short internodes. A reassessment about the pattern of character change for sound production is presented and discussed.     

Analysis of the Distribution of Bootstrap Tree Lengths Using the Maximum Parsimony Method

The bootstrap is an important tool for estimating the confidence interval of monophyletic groups within phylogenies. Although bootstrap analyses are used in most evolutionary studies, there is no clear consensus as how best to interpret bootstrap probability values. To study further the bootstrap method, nine small subunit ribosomal DNA (SSU rDNA) data sets were submitted to bootstrapped maximum parsimony (MP) analyses using unweighted and weighted sequence positions. Analyses of the lengths (i.e., parsimony steps) of the bootstrap trees show that the shape and mean of the bootstrap tree distribution may provide important insights into the evolutionary signal within the sequence data. With complex phylogenies containing nodes defined by short internal branches (multifurcations), the mean of the bootstrap tree distribution may differ by 2 standard deviations from the length of the best tree found from the original data set. Weighting sequence positions significantly increases the bootstrap values at internal nodes. There may, however, be strong bootstrap support for conflicting species groupings among different data sets. This phenomenon appears to result from a correlation between the topology of the tree used to create the weights and the topology of the bootstrap consensus tree inferred from the MP analysis of these weighted data. The analyses also show that characteristics of the bootstrap tree distribution (e.g., skewness) may be used to choose between alternative weighting schemes for phylogenetic analyses.     

Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

Ongoing hybridization and retained ancestral polymorphism in rapidly radiating lineages could mask recent cladogenetic events. This presents a challenge for the application of molecular phylogenetic methods to resolve differences between closely related taxa. We reanalyzed published genotyping‐by‐sequencing (GBS) data to infer the phylogeny of four species within the Ophrys sphegodes complex, a recently radiated clade of orchids. We used different data filtering approaches to detect different signals contained in the dataset generated by GBS and estimated their effects on maximum likelihood trees, global F_ST and bootstrap support values. We obtained a maximum likelihood tree with high bootstrap support, separating the species by using a large dataset based on loci shared by at least 30% of accessions. Bootstrap and F_ST values progressively decreased when filtering for loci shared by a higher number of accessions. However, when filtering more stringently to retain homozygous and organellar loci, we identified two main clades. These clades group individuals independently from their a priori species assignment, but were associated with two organellar haplotype clusters. We infer that a less stringent filtering preferentially selects for rapidly evolving lineage‐specific loci, which might better delimit lineages. In contrast, when using homozygous/organellar DNA loci the signature of a putative hybridization event in the lineage prevails over the most recent phylogenetic signal. These results show that using differing filtering strategies on GBS data could dissect the organellar and nuclear DNA phylogenetic signal and yield novel insights into relationships between closely related species.     

Species tree inference in a recent radiation of orioles (Genus Icterus): multiple markers and methods reveal cytonuclear discordance in the northern oriole group

Recent computational advances provide novel opportunities to infer species trees based on multiple independent loci. Thus, single gene trees no longer need suffice as proxies for species phylogenies. Several methods have been developed to deal with the challenges posed by incomplete and stochastic lineage sorting. In this study, we employed four Bayesian methods to infer the phylogeny of a clade of 11 recently diverged oriole species within the genus Icterus. We obtained well-resolved and mostly congruent phylogenies using a set of seven unlinked nuclear intron loci and sampling multiple individuals per species. Most notably, Bayesian concordance analysis generally agreed well with concatenation; the two methods agreed fully on eight of nine nodes. The coalescent-based method ^∗BEAST further supported six of these eight nodes. The fourth method used, BEST, failed to converge despite exhaustive efforts to optimize the tree search. Overall, the results obtained by new species tree methods and concatenation generally corroborate our findings from previous analyses and data sets. However, we found striking disagreement between mitochondrial and nuclear DNA involving relationships within the northern oriole group. Our results highlight the danger of reliance on mtDNA alone for phylogenetic inference. We demonstrate that in spite of low variability and incomplete lineage sorting, multiple nuclear loci can produce largely congruent phylogenies based on multiple species tree methods, even for very closely-related species.     

Genome-scale phylogenetics: inferring the plant tree of life from 18,896 gene trees

Phylogenetic analyses using genome-scale data sets must confront incongruence among gene trees, which in plants is exacerbated by frequent gene duplications and losses. Gene tree parsimony (GTP) is a phylogenetic optimization criterion in which a species tree that minimizes the number of gene duplications induced among a set of gene trees is selected. The run time performance of previous implementations has limited its use on large-scale data sets. We used new software that incorporates recent algorithmic advances to examine the performance of GTP on a plant data set consisting of 18,896 gene trees containing 510,922 protein sequences from 136 plant taxa (giving a combined alignment length of >2.9 million characters). The relationships inferred from the GTP analysis were largely consistent with previous large-scale studies of backbone plant phylogeny and resolved some controversial nodes. The placement of taxa that were present in few gene trees generally varied the most among GTP bootstrap replicates. Excluding these taxa either before or after the GTP analysis revealed high levels of phylogenetic support across plants. The analyses supported magnoliids sister to a eudicot + monocot clade and did not support the eurosid I and II clades. This study presents a nuclear genomic perspective on the broad-scale phylogenic relationships among plants, and it demonstrates that nuclear genes with a history of duplication and loss can be phylogenetically informative for resolving the plant tree of life.     

COMBINED AND SEPARATE ANALYSES OF MORPHOLOGICAL AND MOLECULAR DATA IN THE PLANT FAMILY RUBIACEAE

Abstract— The Rubiaceae are one of the largest of the families of angiosperms, with over 10000 species. The tribal and subfamilial classification is provisional due to the lack of phylogenetic hypotheses. The present study of the Rubiaceae is based on 33 genera and three data sets, one morphological and two molecular from chloroplast DNA, restriction sites andrbcL sequences. There is much congruence between the morphological and the molecular data sets, but also conflict. For parsimony reasons, the best phylogenetic hypothesis is a tree based on an analysis of the combined data sets. The so-called “total evidence” criterion for the combined analysis is simply a reiteration of the principle of parsimony. In this particular study, the classification would be almost the same even if based on the separate analyses instead of the combined. Despite the inapplicability of consensus trees or trees from separate analyses for phylogenetic hypotheses and classification, separate analyses may provide important information. It is the best way to reveal conflicts between different data sets. Knowledge of the conflicts can promote further detailed investigation in order to improve understanding of characters and phylogenetic hypotheses. In this study, the tribe Vanguerieae provides such an example; morphological data support a position in the subfamily Cinchonoideae, but DNA and a tree based on the combined data support a position in subfamily Ixoroideae. The tribe's position in the morphological tree is probably due to missing information concerning the correct pollen presentation system. Bootstrap fractions and K. Bremer's branch support values are used to evaluate the stability of particular nodes in the trees. Interestingly these values are not always correlated, e.g. in the morphological tree, the node with the highest branch support value has very low bootstrap fraction. The reasons for these differences are unclear, but large differences are presumably more likely to occur on short branches.     

A phylogenetic analysis of Coelopidae (Diptera) based on morphological and DNA sequence data

The phylogenetic relationships of 22 species of Coelopidae are reconstructed based on a data matrix consisting of morphological and DNA sequence characters (16S rDNA, EF-1alpha). Optimal gap and transversion costs are determined via a sensitivity analysis and both equal weighting and a transversion cost of 2 are found to perform best based on taxonomic congruence, character incongruence, and tree support. The preferred phylogenetic hypothesis is fully resolved and well-supported by jackknife, bootstrap, and Bremer support values, but it is in conflict with the cladogram based on morphological characters alone. Most notably, the Coelopidae and the genus Coelopa are not monophyletic. However, partitioned Bremer Support and an analysis of node stability under different gap and transversion costs reveal that the critical clades rendering these taxa non-monophyletic are poorly supported. Furthermore, the monophyly of Coelopidae and Coelopa is not rejected in analyses using 16S rDNA that was manually aligned. The resolution of the tree based on this reduced data sets is, however, lower than for the tree based on the full data sets. Partitioned Bremer support values reveal that 16S rDNA characters provide the largest amount of tree support, but the support values are heavily dependent on analysis conditions. Problems with direct comparison of branch support values for trees derived using fixed alignments with those obtained under optimization alignment are discussed. Biogeographic history and available behavioral and genetic data are also discussed in light of this first cladogram for Coelopidae based on a quantitative phylogenetic analysis.     

Phylogenetic relationships of bolitoglossine salamanders: a demonstration of the effects of combining morphological and molecular data sets

We analyzed sequence data for 555 bp of the mitochondrial gene cytochrome b in plethodontid salamanders, taken from 18 ingroup (tribe Bolitoglossini) and 4 outgroup (tribe Plethodontini) taxa. There were 257 variable sites, of which 219 were phylogenetically informative. Sequence differences among taxa exceeded 20%, and there were up to 15% amino acid differences among the sequences. We also analyzed 37 morphological (including karyological) characters, taken from the literature. Data were analyzed separately and then combined using parsimony and likelihood approaches. There is little conflict between the morphological and DNA data, and that which occurs is at nodes that are weakly supported by one or both of the data sets. Treated separately, the morphological and DNA data provide strong support for some nodes but not for others. The combined data act synergistically so that good support is obtained for nearly all of the nodes in the tree. Recent divergences are supported by silent transitions, and older divergences are supported by a combination of morphological, karyological, DNA transversion, and amino acid changes. Eliminating silent changes from the DNA data improves the consistency index and improves some bootstrap and decay index values for several deeper branches in the tree. However, the combined data set with all characters included provides a better supported tree overall. Maximum likelihood and parsimony with all of the data give not only the same topology but also remarkably similar branch lengths. Results of this analysis support the monopoly of the supergenera Hydromantes and Batrachoseps, and of a sister group relationship of Batrachoseps and the supergenus Bolitoglossa (represented in this study one species of the genus Bolitoglossa).      

Molecular phylogeny and plumage evolution in gulls (Larini)

We used DNA sequence data of the mitochondrial control region and cytochrome b gene to assess phylogenetic relationships among 32 gull species and two outgroup representatives. We tentatively estimated divergence times from transversional substitutions calibrated against DNA–DNA hybridization data. Several strongly supported species groups are identified, but the relationships between these species groups and the rooting of the gull tree remain unresolved. Geographical range extension appears as a factor of speciation, but several related, well‐differentiated species seem to have evolved within comparatively restricted areas. Some plumage characters used in the past for delimiting species groups appear inappropriate. The dark hooded species, for instance, do not constitute a natural assemblage. Molecular data also allowed the identification of several striking plumage convergences that had obscured the true relationships between gull species until now. For example, the dark tropical gulls analysed here each belong to totally different clades and are independent examples of convergent plumage evolution under common environmental constraints. The reverse situation also happened, with two arctic‐distributed species, the ivory gull (Pagophila eburnea) and the Sabine’s gull (Xema sabini), appearing as sister taxa despite completely different plumage features. Molecular data have thus significantly improved our understanding of gull evolution.     

Chloroplast DNA diversity in the genusRubus (Rosaceae) revealed by Southern hybridization

The variability in chloroplast DNA type of 20Rubus genotypes was examined by Southern hybridization. DNA extracted from theRubus accessions was digested with two restriction enzymes (EcoRI and EcoRV) and heterologous chloroplast DNA sequences from barley and pea were used as probes to detectRubus chloroplast DNA sequences on Southern blots ofRubus total DNA. Restriction fragment length polymorphism was detected and a total of 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and is in general agreement with information based on morphological criteria. However, the Japanese red raspberryR. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subg.Idaeobatus. Furthermore, the molecular approach provides a quantitative estimate of the relationship between species that is difficult to obtain from morphological data. In order to complement the chloroplast DNA information a ribosomal DNA probe was also included in the analysis and provided further information on the phylogenetic relationships withinRubus.     

The Relationship between the Number of Loci and the Statistical Support for the Topology of UPGMA Trees Obtained from Genetic Distance Data

An analysis of the relationship between the number of loci utilized in an electrophoretic study of genetic relationships and the statistical support for the topology of UPGMA trees is reported for two published data sets. These are Highton and Larson (Syst. Zool.28: 579-599, 1979), an analysis of the relationships of 28 species of plethodonine salamanders, and Hedges (Syst. Zool., 35: 1-21, 1986), a similar study of 30 taxa of Holarctic hylid frogs. As the number of loci increases, the statistical support for the topology at each node in UPGMA trees was determined by both the bootstrap and jackknife methods. The results show that the bootstrap and jackknife probabilities supporting the topology at some nodes of UPGMA trees increase as the number of loci utilized in a study is increased, as expected for nodes that have groupings that reflect phylogenetic relationships. The pattern of increase varies and is especially rapid in the case of groups with no close relatives. At nodes that likely do not represent correct phylogenetic relationships, the bootstrap probabilities do not increase and often decline with the addition of more loci.     

Phylogeny of the Heelwalkers (Insecta: Mantophasmatodea) based on mtDNA sequences, with evidence for additional taxa in South Africa

We examined the phylogeny of Mantophasmatodea from southern Africa (South Africa, Namibia) using approx. 1300 bp of mitochondrial DNA sequence data from the genes encoding COI and 16S. The taxon sample comprised multiple specimens from eight described species (Namaquaphasma ookiepense, Austrophasma rawsonvillense, A. caledonense, A. gansbaaiense, Lobatophasma redelinghuysense, Hemilobophasma montaguense, Karoophasma botterkloofense, K. biedouwense) and four undescribed species of Austrophasmatidae; three specimens of Sclerophasma paresisense (Mantophasmatidae); and two specimens of Praedatophasma maraisi and one of Tyrannophasma gladiator (not yet convincingly assigned to any family). For outgroup comparison a broad selection from hemimetabolous insect orders was included. Equally weighted parsimony analyses of the combined COI+16S data sets with gaps in 16S scored as a fifth character state supported Austrophasmatidae and all species and genera of Mantophasmatodea as being monophyletic. Most species were highly supported with 98-100% bootstrap/7-39 Bremer support (BS), but K. biedouwense had moderate support (87/4) and A. caledonense low support (70/1). Mantophasmatodea, Austrophasmatidae, and a clade Tyrannophasma gladiator+Praedatophasma maraisi were all strongly supported (99-100/12-25), while relationships among the two latter clades and Mantophasmatidae remain ambiguous. Concerning the relationships among genera of Austrophasmatidae, support values are moderately high for some nodes, but not significant for others. We additionally calculated the partitioned BS values of COI and 16S for all nodes in the strict consensus of the combined tree. COI and 16S are highly congruent at the species level as well as at the base of Mantophasmatodea, but congruence is poor for most intergeneric relationships. In forthcoming studies, deeper relationships in the order should be additionally explored by nuclear genes, such as 18S and 28S, for a reduced sample of specimens.