Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Differentiation of staphylococcal species and strains by ribosomal RNA gene restriction patterns

Staphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

TRIBAL PLACEMENT OF MARSHALLIA (ASTERACEAE) USING CHLOROPLAST DNA RESTRICTION SITE MAPPING

The examination of morphological traits has failed to resolve the tribal placement of Marshallia. Suggested relationships for this anomalous genus have, at various times, included Eupatorieae, Heliantheae, Vemonieae, and Inuleae. Chloroplast DNA restriction site mapping, using Bamadesiinae (Mutisieae) as the outgroup, revealed 981 restriction site mutations, 332 of which were phylogenetically informative, for 60 genera representing 15 tribes of Asteraceae. Wagner parsimony produced 36 equally parsimonious tress of 729 steps, and Dollo parsimony produced 34 equally parsimonious trees of 759 steps. Monophyletic groups, resulting from the Wagner analysis, were further tested with the bootstrap method. The placement of Marshallia in the Heliantheae-Tageteae-Coreopsideae-Eupatorieae complex was consistent for all trees produced. Tageteae and Coreopsideae form the sister group to paraphyletic Heliantheae, with Marshallia sharing its most recent common ancestor with Galinsoga, Palafoxia, and Bahia. The Eupatorieae form a monophyletic clade that is derived from helianthoid ancestors.     

Molecular differentiation of the microgastrine species commonly found in paddy fields from Southeast Asia,with additional data on their phylogeny （Hymenoptera：Braconidae）

Partial DNA sequences of three genes, that is, mitochondrial large ribosomal subunit (16S), nuclear large ribosomal subunit (28S D2) and mitochondrial NADH1 dehydrogenase (NADH1) gene, were sequenced from different microgas trine species(Braconidae: Microgastrinae) collected fresh from paddy fields. The DNA sequences were used to determine the extent of sequence variation among species in order to evaluate the specific status of each species. Cladistic analysis was also used to infer a phylogenetic relationship among these species. The results showed that sequence divergence among species of the same genus Cotesia was much lower than those among different genera, such as Cotesia, Exoryza and Apanteles; the sequence similarity of 16S rDNA and NADH 1 genes between Cotesia sp. and C. chilonis was higher than that between C. sp. and C. ruficrus.Phylogenetic analyses suggested that four species of Cotesia were always grouped in the same clade regardless of using different analysis methods; Cotesia sp. and C. chilonis are more closely related to each other than to C. ruficrus, different from previous morphological results. Additionally, sequence analyses indicated that NADH1 gene has more parsimony informative characters than 28S rDNA D2 and 16S rDNA at the species-level analysis,indicating that NADH1 gene might be a useful marker for species-level analysis.     

Chloroplast DNA restriction site variation and phylogeny of the Berberidaceae

Comparative restriction site mapping of the chloroplast genome was performed to examine phylogenetic relationships among 27 species representing 16 genera of the Berberidaceae and two outgroups. Chloroplast genomes of the species included in this study showed no major structural rearrangements (i.e., they are collinear to tobacco cpDNA) except for the extension of the inverted repeat in species of Berberis and Mahonia. Excluding several regions that exhibited severe length variation, a total of 501 phylogenetically informative sites was mapped for ten restriction enzymes. The strict consensus tree of 14 equally parsimonious trees indicated that some berberidaceous genera (Berberis, Mahonia, Diphylleia) are not monophyletic. To explore phylogenetic utility of different parsimony methods phylogenetic trees were generated using Wagner, Dollo, and weighted parsimony for a reduced data set that included 18 species. One of the most significant results was the recognition of the four chromosomal groups, which were strongly supported regardless of the parsimony method used. The most notable difference among the trees produced by the three parsimony methods was the relationships among the four chromosomal groups. The cpDNA trees also strongly supported a close relationship of several generic pairs (e.g., Berberis-Mahonia, Epimedium-Vancouveria, etc.). Maximum likelihood values were computed for the four different tree topologies of the chromosomal groups, two Wagner, one Dollo, and one weighted topology. The results indicate that the weighted tree has the highest likelihood value. The lowest likelihood value was obtained for the Dollo tree, which had the highest bootstrap and decay values. Separate analyses using only the Inverted Repeat (IR) region resulted in a tree that is identical to the weighted tree. Poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae indicate that they may have radiated from an ancestral stock in a relatively short evolutionary time.     

Phylogenetic signal in AFLP data sets

AFLP markers provide a potential source of phylogenetic information for molecular systematic studies. However, there are properties of restriction fragment data that limit phylogenetic interpretation of AFLPs. These are (a) possible nonindependence of fragments, (b) problems of homology assignment of fragments, (c) asymmetry in the probability of losing and gaining fragments, and (d) problems in distinguishing heterozygote from homozygote bands. In the present study, AFLP data sets of Lactuca s.l. were examined for the presence of phylogenetic signal. An indication of this signal was provided by carrying out tree length distribution skewness (g1) tests, permutation tail probability (PTP) tests, and relative apparent synapomorphy analysis (RASA). A measure of the support for internal branches in the optimal parsimony tree (MPT) was made using bootstrap, jackknife, and decay analysis. Finally, the extent of congruence in MPTs for AFLP and internal transcribed spacer (ITS)-1 data sets for the same taxa was made using the partition homogeneity test (PHT) and the Templeton test. These analytical studies suggested the presence of phylogenetic signal in the AFLP data sets, although some incongruence was found between AFLP and ITS MPTs. An extensive literature survey undertaken indicated that authors report a general congruence of AFLP and ITS tree topologies across a wide range of taxonomic groups, suggesting that the present results and conclusions have a general bearing. In these earlier studies and those for Lactuca s.l., AFLP markers have been found to be informative at somewhat lower taxonomic levels than ITS sequences. Tentative estimates are suggested for the levels of ITS sequence divergence over which AFLP profiles are likely to be phylogenetically informative.     

Study of the evolutionary relationships among Limonium species (Plumbaginaceae) using nuclear and cytoplasmic molecular markers

The genus Limonium, due to the patchiness of the natural habitats of its species as well as the high frequency of hybridization and polyploidy and the possibility of reproduction by apomixis, provides an example of all the principal mechanisms of rapid speciation of plants. As an initial study of evolution in this genus, we have analyzed intra- and interspecific variability in 17 species from section Limonium, the largest in the genus, based on RFLPs of cpDNA and nuclear rDNA ITS sequences. In the cpDNA analysis, 21 restriction enzymes were used, resulting in 779 fragments, 490 of which were variable and 339 parsimony informative. L. furfuraceum exhibited two relatively divergent cpDNA haplotypes. The relationships found among the species based on cpDNA restriction fragments were coincident using different methods of phylogenetic analysis. Due to the presumed reticulate evolution in the genus Limonium, the comparison of these results with data from the nuclear DNA was necessary; ITS sequences were analyzed. The final alignment contained 488 characters, of which 198 were variable and 156 parsimony informative. Two relatively divergent ITS types were present at the intraindividual level in L. delicatulum, a triploid species. Each type was related to ITS from different groups of diploid Limonium species, one with a base haploid chromosome number n = 8 (represented by L. cossonianum) and the other with n = 9 (represented by L. minutum). The different phylogenetic inference methods used for the analysis of ITS sequences rendered very similar topologies. In general, the relationships among the species studied were coincident with those obtained with the chloroplast genome. Both nuclear and cytoplasmic markers support the polyphyly of section Limonium, with at least two species, L. narbonense and L. vulgare, clearly divergent from the rest. Moreover, the remaining subsections into which section Limonium is currently divided seem to be artificial.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

Genetic diversity and phylogenetic relationships in Vigna Savi germplasm revealed by DNA amplification fingerprinting.

The pantropical genus Vigna (Leguminosae) comprises 7 cultivated species that are adapted to a wide range of extreme agroclimatic conditions. Few data are available on the relationships among these cultivated species or on their importance as sources of resistance against biotic and abiotic stresses. Therefore, we optimized DNA amplification fingerprinting (DAF) to estimate the genetic diversity within, and genetic relationships among, a representative core collection of cowpea, as compared with 16 accessions representing cultivars from 6 Vigna species. A set of 26 primers was selected from 262 tested random primers and used for the characterization of 85 Vigna accessions (6 V. angularis, 4 each of V. mungo and V. radiata, 2 V. umbellata, 1 V. aconitifolia, and 68 V. unguiculata), with Phaseolus vulgaris subsp. vulgaris as outgroup. A total of 212 polymorphic bands were used for maximum parsimony analysis. Our results clearly distinguished Brazilian from African V. unguiculata genotypes. At the species level, V. angularis was the most related and V. radiata the most divergent species relative to V. unguiculata. DAF markers were also informative at the intraspecific level, detecting a large diversity between cowpea cultivars. The implications of the presented results for cowpea breeding programs are discussed.     

Phylogeny of Melaleuca, Callistemon, and Related Genera of the Beaufortia Suballiance (Myrtaceae) Based on 5S and ITS-1 Spacer Regions of nrDNA

A phylogenetic analysis of genera within the informal suballiance Beaufortia (family Myrtaceae), largely endemic to Australia and New Caledonia, is presented based on separate and combined data sets for 5S and ITS-1 spacer regions of nuclear ribosomal DNA. The two sets were not in conflict but the 5S data set was more informative. Data were analysed using conventional parsimony, jackknife parsimony, and three-item parsimony analyses. Three-item analysis gave more resolved trees than conventional parsimony analysis. The Beaufortia suballiance includes two major clades, with all Australian representatives of Callistemon (shown to be monophyletic) and most Australian representatives of Melaleuca forming one of these. The sister clade comprises a well-defined group of endemic New Caledonian taxa (classified as Callistemon and Melaleuca ), some Australian species of Melaleuca , a clade including the Western Australia/Northern Territory genera Beaufortia, Lamarchea , and Regelia , and a clade including the south-west Western Australian genera Calothamnus, Eremaea, Conothamnus , and Phymatocarpus . All molecular analyses sup port the monophyly of Conothamnus and of Regelia , genera for which a number of species were included. Three-item analysis of the combined data set supports the monophyly of Beaufortia . The findings have implications for both taxonomy and biogeography.     

Phylogenetic relationships of the pigeonpea (Cajanus cajan) based on nuclear restriction fragment length polymorphisms.

Nuclear restriction fragment length polymorphisms (RFLPs) were used to determine phylogenetic relationships in the genus Cajanus using 15 random genomic probes and six restriction enzymes. Twenty-four accessions representing 12 species of four genera (Cajanus, Dunbaria, Eriosema, and Rhynchosia) were examined to determine phylogenetic relationships in the genus Cajanus. Eriosema parviflorum was selected as the out-group. Sufficient RFLP polymorphisms were detected among species to resolve in-group taxa into distinct clusters. Topologies of trees from parsimony and similarity matrix analyses were similar but not identical, and clustering patterns agreed broadly with published phylogenies based on seed protein data and, to a lesser extent, data from cytology and breeding experiments. Accessions of cultivated C. cajan shared more DNA fragments with C. scarabaeoides than with C. cajanifolia. Inconsistencies in taxonomic relationships based on data from morphology, cytology, crossability, and RFLPs are discussed.     

Development of chloroplast genomic resources for Cynara

In this study, new chloroplast (cp) resources were developed for the genus Cynara, using whole cp genomes from 20 genotypes, by means of high‐throughput sequencing technologies. Our target species included seven globe artichokes, two cultivated cardoons, eight wild artichokes, and three other wild Cynara species (C. baetica, C. cornigera and C. syriaca). One complete cp genome was isolated using short reads from a whole‐genome sequencing project, while the others were obtained by means of long‐range PCR, for which primer pairs are provided here. A de novo assembly strategy combined with a reference‐based assembly allowed us to reconstruct each cp genome. Comparative analyses among the newly sequenced genotypes and two additional Cynara cp genomes (‘Brindisino’ artichoke and C. humilis) retrieved from public databases revealed 126 parsimony informative characters and 258 singletons in Cynara, for a total of 384 variable characters. Thirty‐nine SSR loci and 34 other INDEL events were detected. After data analysis, 37 primer pairs for SSR amplification were designed, and these molecular markers were subsequently validated in our Cynara genotypes. Phylogenetic analysis based on all cp variable characters provided the best resolution when compared to what was observed using only parsimony informative characters, or only short ‘variable’ cp regions. The evaluation of the molecular resources obtained from this study led us to support the ‘super‐barcode’ theory and consider the total cp sequence of Cynara as a reliable and valuable molecular marker for exploring species diversity and examining variation below the species level.     

CHLOROPLAST DNA RESTRICTION SITE VARIATION IN THE FERN GENUS PELLAEA: PHYLOGENETIC RELATIONSHIPS OF THE PELLAEA GLABELLA COMPLEX

The cheilanthoid ferns have long resisted efforts to circumscribe well-defined, phylogenetically natural generic and infrageneric groups, presumably because of homoplastic morphologies associated with their xeric habitats. This cladistic analysis of phylogenetically informative chloroplast DNA restriction site data from 14 enzymes and seven taxa in the cheilanthoid genus Pellaea provides new insights into the phylogenetic relationships of the P. glabella complex. It also assesses the congruence of results based on restriction site data at inter- and intraspecific levels in these sexually and apogamously reproducing ferns with those of earlier morphological and isozyme analyses of the same group. Wagner parsimony yielded a single most parsimonious tree of 187 steps and 11% homoplasy, based on a data matrix of 166 restriction sites of which 66 were phylogenetically informative. Phylogenetic analysis based on user-defined stepmatrix character-state weighting of site gains over losses produced an identical single most parsimonious tree. Dollo parsimony yielded two most parsimonious trees, one of which was topologically identical to the Wagner tree. Specific and infraspecific relationships in the P. glabella complex determined by the completely independent restriction site and isozyme data sets are identical. This lends confidence to the ongoing use of restriction site data in a broader study of Pellaea and other cheilanthoid taxa and to the present conclusions that P. atropurpurea is sister to the P. glabella complex, whereas P. breweri, previously considered the closest relative of this complex, is actually more distantly related to it than are the other taxa in this study.     

Genetic Variation at the Mitochondrial 16S rRNA Gene among Trichiurus lepturus (Teleostei:Trichiuridae) from Various Localities: Preliminary Evidence of a New Species from West Coast of Africa

The taxonomic classification of large head hairtail (Trichiurus lepturus) is controversial because of similar body appearance and silvery coloration. Ten samples of T. lepturus caught off the west coast of Africa and 43 samples of T. lepturus caught off the western Atlantic coast and Indo-Pacific waters were used for sequence comparison of mitochondrial DNA encoded partial 16S ribosomal RNA(16S rRNA) gene. Ten samples of T. japonicus (obtained from various parts of southern Japan) were also included in the comparative analysis. For the 509 bp sequence determined, 58 sites were variable, of which 53 were parsimony informative. Phylogenetic analyses using maximum parsimony, maximum likelihood and Bayesian algorithm with Eupleurogrammus muticus as outgroup, showed that the haplotypes of T. lepturus obtained from the West coast of Africa, Indo-Pacific and Western Atlantic coast constituted clearly distinct and well supported lineages without any sharing and overlapping between them. Previous morphological analyses and this genetic study strongly indicate that the morphotypes of T. lepturus obtained from the West African coast are genetically distinct and probably represents a separate species.     

Phylogeny of Australian species of flatheads (Teleostei, Platycephalidae) as determined by allozyme electrophoresis

Phylogenetic relationships of 24 taxa of Platycephalid fish were examined for up to 40 loci using starch and polyacrylarnide gel electrophoresis. Over 400 different enzyme mobilities were scored. The extent of homoplasy was estimated to be between 5 and 12%. Polymorphism at the 1% level was observed at 26 of the 40 loci. The importance of shared polymorphic alleles in cladistic analysis was endorsed by the different results obtained from multistate and binary coding of alleles. Many loci were shown to contain phylogenetically informative ancestral alleles which appear irregularly in extant species. The polymorphism parsimony method provided the most informative results. Genetic similarities between endemic Australian species from each family stem provide the basis for a major revision of subfamilies and genera within the family.     

Phylogenetic systematics of New WorldIpomoea (Convolvulaceae) based on chloroplast DNA restriction site variation

Chloroplast DNA restriction site variation was studied in 31 New World species ofIpomoea, representing a majority of the New World sections and series within the genus. Using 14 endonucleases, a total of 124 phylogenetically informative restriction sites was detected. Dollo parsimony, Wagner parsimony, and bootstrap methods were employed to construct phylogenetic trees and evaluate confidence intervals of monophyletic groups. With a few exceptions, groups circumscribed on the basis of morphological variation are in agreement with groupings based on restriction site variation. Relationships between subgeneric groupings, however, disagree substantially with those proposed in the past. Although conflicting hypotheses for some intersectional relationships are not presently resolvable, cpDNA restriction site analyses propose the following refinements of existing classification schemes.Ipomoea ser.Setosae is divided into distantly related groups, as is sect.Pharbitis. SeriesTyrianthinae, a proposed segregate of sect.Pharbitis, is associated with sect.Calonyction and the Tricolor complex (subg.Quamoclit).Ipomoea sect.Batatas is segregated from other herbaceous groups of the heterogeneous subg.Quamoclit sensu lato and aligned as a derivative ofI. setosa, subg.Eriospermum. To test for homology of key characters weighted in traditional schemes, morphological features were studied with respect to their distribution on lineages defined by restriction site data. Characters such as setose sepals, foliose-pubescent sepals, and erect growth habit, among others, are interpreted as having multiple origins, while 3-locular ovaries, 4-locular ovaries, and long-haired seeds have evolved only once.     

VARIATION IN MITOCHONDRIAL DNA OF AEDES SPECIES (DIPTERA: CULICIDAE)

Relationships among seven species of the Aedes scutellaris subgroup and four of the Aedes albopictus subgroup were determined using restriction fragment analysis of mtDNA. Considerable polymorphism was found among species, with an average proportion of 0.338 restriction fragments being shared among them. Fitch-Margoliash, Wagner parsimony and UPGMA analyses revealed that the phylogeny based on mtDNA was only partially congruent with that based on allozymes and morphology. All three analyses, however, suggested that three species pairs, A. flavopictus-A. pseudalbopictus, A. pseudoscutellaris-A. riversi, and A. albopictus-A. polynesiensis were monophyletic with regard to their mtDNA. The results indicated that although the species in the A. scutellaris and the A. albopictus subgroups do not exhibit significant morphological divergence, their mtDNA has diverged extensively.