Chloroplast Genome Types Research Articles

Comparative analysis of 84 chloroplast genomes of Tylosema esculentum reveals two distinct cytotypes.

Tylosema esculentum (marama bean) is an important orphan legume from southern Africa that has long been considered to have the potential to be domesticated as a crop. The chloroplast genomes of 84 marama samples collected from various geographical locations in Namibia and Pretoria were compared in this study. The cp genomes were analyzed for diversity, including SNPs, indels, structural alterations, and heteroplasmy. The marama cp genomes ranged in length from 161,537 bp to 161,580 bp and contained the same sets of genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The genes rpoC2 and rpoB, and the intergenic spacers trnT-trnL and ndhG-ndhI were found to be more diverse than other regions of the marama plastome. 15 haplotypes were found to be divided into two groups, differing at 122 loci and at a 230 bp inversion. One type appears to have greater variability within the major genome present, and variations amongst individuals with this type of chloroplast genome seems to be distributed within specific geographic regions but with very limited sampling for some regions. However, deep sequencing has identified that within most of the individuals, both types of chloroplast genomes are present, albeit one is generally at a very low frequency. The inheritance of this complex of chloroplast genomes appears to be fairly constant, providing a conundrum of how the two genomes co-exist and are propagated through generations. The possible consequences for adaptation to the harsh environment in which T. esculentum survives are considered. The results pave the way for marama variety identification, as well as for understanding the origin and evolution of the bean.

Hybrid Speciation and Introgression Both Underlie the Genetic Structures and Evolutionary Relationships of Three Morphologically Distinct Species of Lilium (Liliaceae) Forming a Hybrid Zone Along an Elevational Gradient.

We studied hybrid interactions of Lilium meleagrinum, Lilium gongshanense, and Lilium saluenense using an integrative approach combining population genetics, fieldwork, and phenological research. These three species occur along an elevational gradient, with L. meleagrinum occurring at lower elevations, L. saluenense at higher elevations, and L. gongshanense between them. The species show strong morphological differentiation despite there being no clear environmental barriers to gene flow among them. Lilium gongshanense is likely to have a hybrid origin based on our prior work, but its progenitors remain uncertain. We sought to determine whether gene flow occurs among these three parapatric species, and, if so, whether L. gongshanense is a hybrid of L. meleagrinum and/or L. saluenense. We analyzed data from multiple chloroplast genes and spacers, nuclear internal transcribed spacer (ITS), and 18 nuclear Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) microsatellites for accessions of the three species representing dense population-level sampling. We also inferred phenology by examining species in the field and using herbarium specimens. We found that there are only two types of chloroplast genomes shared among the three species and that L. gongshanense forms two distinct groups with closest links to other species of Lilium based on ITS. Taken together, L. gongshanense is unlikely to be a hybrid species resulting from a cross between L. meleagrinum and L. saluenense, but gene flow is occurring among the three species. The gene flow is likely to be rare according to evidence from all molecular datasets, and this is corroborated by detection of only one putative hybrid individual in the field and asynchronous phenology. We suspect that the rarity of hybridization events among the species facilitates their continued genetic separation.

A Phylogenetic Analysis of Chloroplast Genomes Elucidates the Relationships of the Six Economically Important Brassica Species Comprising the Triangle of U.

The Brassica genus comprises many economically important worldwide cultivated crops. The well-established model of the Brassica genus, U’s triangle, consists of three basic diploid plant species (Brassica rapa, Brassica oleracea, and Brassica nigra) and three amphidiploid species (Brassica napus, Brassica juncea, and Brassica carinata) that arose through interspecific hybridizations. Despite being extensively studied because of its commercial relevance, several aspects of the origin of the Brassica species and the relationships within and among these six species still remain open questions. Here, we successfully de novo assembled 60 complete chloroplast genomes of Brassica genotypes of all six species. A complete map of the single nucleotide variants and insertions and deletions in the chloroplast genomes of different Brassica species was produced. The chloroplast genome consists of a Large and a Small Single Copy (LSC and SSC) region between two inverted repeats, and while these regions of chloroplast genomes have very different molecular evolutionary rates, phylogenetic analyses of different regions yielded no contradicting topologies and separated the Brassica genus into four clades. B. carinata and B. juncea share their chloroplast genome with one of their hybridization donors B. nigra and B. rapa, respectively, which fits the U model. B. rapa, surprisingly, shows evidence of two types of chloroplast genomes, with one type specific to some Italian broccoletto accessions. B. napus clearly has evidence for two independent hybridization events, as it contains either B. rapa chloroplast genomes. The divergence estimation suggests that B. nigra and B. carinata diverged from the main Brassica clade 13.7 million years ago (Mya), while B. rapa and B. oleracea diverged at 2.18 Mya. The use of the complete chloroplast DNA sequence not only provides insights into comparative genome analysis but also paves the way for a better understanding of the phylogenetic relationships within the Brassica genus.

Molecular Analysis of the Genetic Diversity of Chinese Hami Melon and Its Relationship to the Melon Germplasm from Central and South Asia

Chinese Hami melon consists of the varieties cassaba, chandalak, ameri, and zard. To show their genetic diversity, 120 melon accessions, including 24 accessions of Hami melon, were analyzed using molecular markers of nuclear and cytoplasmic genomes. All Hami melon accessions were classified as the large-seed type with seed length longer than 9 mm, like US and Spanish Inodorus melon. Conomon accessions grown in east China were all the small-seed type. Both large- and small-seed types were in landraces from Iran, Afghanistan, Pakistan, and Central Asia. Analysis of an SNP in the PS-ID region (Rpl16-Rpl14) and size polymorphism of ccSSR7 showed that the melon accessions consisted of three chloroplast genome types, that is, maternal lineages. Hami melon accessions were T/338 bp type, which differed from Spanish melon and US Honey Dew (T/333 bp type), indicating a different maternal lineage within group Inodorus. The gene diversity (D), calculated from random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) polymorphism, was 0.476 in 120 melon accessions; the largest diversity was in Central Asian accessions (D = 0.377) but was low for Hami melon accessions (D = 0.243), even though Hami melon has diverse morphological traits, earliness, and shelf life. Reflecting such small genetic diversity, Hami melon accessions of vars. ameri and zard were grouped into cluster II, except for one accession, by the unweighted pair group method and the arithmetic mean (UPGMA) cluster analysis. Variety chandalak with distinct characters, such as early maturing and poor shelf life, was assigned to clusters IV and VI, indicating inter-varietal genetic differentiation within Hami melon. Three accessions from Turkmenistan and Afghanistan, with large seeds and T/338 bp type of chloroplast genome, were classified as cluster II with Hami melon accessions of vars. ameri and zard. We therefore concluded that Hami melon may have been transmitted from the west. The small-seed type melon of group Conomon grown in east China may have been introduced into China independently of Hami melon, because it had the A/338 bp type of the chloroplast genome and was clustered distantly from Hami melon according to nuclear genome analysis.

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species.

Phylogenetic Relationship in the Genus Rosa Based on the Restriction Enzyme Analysis of the Chloroplast DNA.

To plot the phylogenetic relationship within the genus Rosa, we compared the restriction sites of chloroplast DNAs among 32 species. With the use of 14 restriction endonucleases, restriction fragment patterns of cpDNAs were obtained from 32 lines of Rosa species and compared with each other. Three hundred thirty-five restriction sites were surveyed. Thirty-three sites were phylogenetically informative. Among the chloroplast genome types, 18 primary groups are formed. The majority rule consensus tree established by the Wagner parsimony method indicated that the species in sections Synstylase and Indica, and sections Carolinae and Cinnamomeae belong to a monophyletic clade, which agrees with the morphological classification. Sections Carolinae, Cinnamomeae, and Pimpinelifoliae lie distantly from the other sections, suggesting that these sections may have differentiated in an early stage of evolution followed by the appearance of other sections. A comparison of the previous classification of Rosa species with that in this study, revealed several discrepancies. R. gigantea, R. moschata, and R. damascena, which appear to be monophyletic, were grouped into one cluster apart from section Gallicanae, to which R. damascena was formerly classified. The present molecular data indicate that the maternal ancestor of R. centifolia 'Japonica' may be R. canina. It is proposed that R. roxburghiin and R. roxburghiin hirtula are separated, based on their differences in cpDNA. Interspecific cpDNA variation is common, a great deal of variability was detected in Rosa. Excessive dependence on one particular cytoplasm may be undesirable, we suggest that the methods described in this paper be used to survey the cytoplasmic base of modern rose cultivars.

Phylogenetic relationships of the mitochondrial genomes in the genus Glycine subgenus Soja.

Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) of wild and cultivated soybeans were analyzed to study their phylogenetic relationships. The observed number of differences in hybridization profiles greatly varied (1-17 patterns) with both the mtDNA probes and the restriction enzymes that were used. A cladistic analysis was conducted based on the RFLP data. In the parsimonious tree, four distinct groups appeared among 20 accessions of the subgenus Soja representing 20 mitochondrial genome types. Common features with regard to geographic distributions in natural populations in East Asia were observed among the mitochondrial genome types of wild soybean that belonged to the same group: one clade consisted of genome types IIg and VIIg that are detected with very low frequencies; another clade consisted of genome types Ic, Id, Ie, and Ik whose distributions are highly biased mainly in Japan. The genome types that are widely distributed in East Asia such as IVa, IVb, and Va were not grouped into the same clade. The mitochondrial genome types IIIb, IVb, and IVc, in which two different chloroplast genome types exist, belonged to the same clade. Possible changes in mitochondrial genomes during the expansion of the distribution of wild soybeans in East Asia were discussed.

Genetic variation and interspecific hybridization among natural populations of zoysiagrasses detected by RFLP analyses of chloroplast and nuclear DNA.

Genetic variations among 17 accessions of zoysiagrasses collected from natural populations in Japan were investigated by RFLP analyses of chloroplast DNA (cpDNA) and nuclear DNA. These accessions were classified into five species based on morphological characteristics: Zoysia japonica, Z. matrella, Z. tenuifolia, Z. sinica, and Z. macrostachya. On the basis of eight kinds of RFLPs in cpDNAs detected across accessions, six chloroplast genome types (types A-F) were identified. Although type-A cpDNA was shared by five accessions of japonica and four accessions of matrella, derivative cpDNAs of type A, which each arose by a mutation, were identified in one accession of japonica (type B) and in two accessions of matrella (type C). One accession of japonica which showed spikelets similar to those of shapes macrostachya, contained type-F cpDNA as did sinica and macrostachya. The two accessions of tenuifolia each showed a specific cpDNA type, i.e. types D and E. Genetic relationships among the 17 accessions were investigated by the RFLP analyses of nuclear DNA with 20 genomic and gene probes. A dendrogram constructed with genetic distances calculated from the RFLP patterns indicated four major groups among them. Six accessions of japonica comprised one group, whereas the one accession of japonica possessing the type-F cpDNA was clustered with macrostachya and sinica. Four accessions of matrella with type A cpDNA constituted another group in the dendrogram, showing a closer relationship to the japonica accessions than to the other two accessions of matrella. The remaining two accessions of matrella and tenuifolia accessions were grouped together. These data indicate that zoysiagrasses distributed in Japan harbor highly genetic variations, and that interspecific hybridization has occurred in natural populations.

Origin and phylogeny of Guinea yams as revealed by RFLP analysis of chloroplast DNA and nuclear ribosomal DNA

The origin and phylogeny of the Guinea yams, consisting of the white yam (Dioscorea rotundata Poir.) and the yellow yam (D. cayenensis Lamk.), has been investigated. Fourteen cultivars of Guinea yams were sampled with 12 accessions from seven wild yam species. A total of 26 accessions were surveyed for restriction fragment length polymorphisms (RFLP) in chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) using seven restriction endonucleases and various heterologous probes. Chloroplast DNA probes covering 80% of the total chloroplast genome revealed nine restriction site changes and one length mutation among the cpDNAs of Guinea yams and their wild relatives. The estimated numbers of nucleotide substitutions per site (d) among these cpDNAs were very low (0.0005-0.0027), indicating a rather recent divergence of this group. On the basis of these ten mutations, five chloroplast genome types (A-E) were recognized. It was revealed that two cultivated species (D. rotundata and D. cayenensis) display the same chloroplast genome type, type A, as the three wild species D. praehensilis, D. liebrechtsiana and D. abyssinica. Chloroplast genome types B, C, D and E were found in D. minutiflora, D. burkilliana, D. smilacifolia and D. togoensis, respectively. Maximum parsimony analysis produced a hypothetical phylogeny of three primary lineages among cpDNAs of Guinea yams and their relatives: the genome type A lineage, the genome type B lineage and one lineage including genome types C, D and E.Using rDNA clones of rice and taro as probes, we detected ribosomal DNA variation, presumably at the intergenic spacer region, in Guinea yams and their wild relatives. The survey of rDNA together with that of cpDNA indicates that D. rotundata (white yam) was domesticated from either D. abyssinica, D. liebrechtsiana or D. praehensilis or their hybrid, and that D. cayenensis (yellow yam) is derived from hybridization between a male plant of either D. burkilliana, D. minutiflora or D. smilacifolia and a female plant of either D. rotundata, D. abyssinica, D. liebrechtsiana or D. praehensilis. We propose that the previous nomenclature of white yam should be retained, D. rotundata Poir. nomen nudum, and that yellow yam should be treated as a variety of D. rotundata, denoted as D. rotundata var. x 'cayenensis'.

Molecular analysis of the hot spot region related to length mutations in wheat chloroplast DNAs. I. Nucleotide divergence of genes and intergenic spacer regions located in the hot spot region.

The nucleotide divergence of chloroplast DNAs around the hot spot region related to length mutation in Triticum (wheat) and Aegilops was analyzed. DNA sequences (ca. 4.5 kbp) of three chloroplast genome types of wheat complex were compared with one another and with the corresponding region of other grasses. The sequences region contained rbcL and psaI, two open reading frames, and a pseudogene, rpl23' (pseudogene for ribosomal protein L23) disrupted by AT-rich intergic spacer regions. The evolution of these genes in the closely related wheat complex is characterized by nonbiased nucleotide substitutions in terms of being synonymous/nonsynonymous, having A-T pressure transitions over transversions, and frequent changes at the third codon position, in contrast with the gene evolution among more distant plant groups where biased nucleotide substitutions have frequently occurred. The sequences of these genes had diverged almost in proportion to taxonomic distance. The sequence of the pseudogene rpl23' changed approximately two times faster than that of the coding region. Sequence comparison between the pseudogene and its protein-coding counterpart revealed different degrees of nucleotide homology in wheat, rice and maize, suggesting that the transposition timing of the pseudogene differed and/or that different rates of gene conversion operated on the pseudogene in the cpDNA of the three plant groups in Gramineae. The intergenic spacer regions diverged approximately ten times faster than the genes. The divergence of wheat from barley, and that from rice are estimated based on the nucleotide similarity to be 1.5, 10 and 40 million years, respectively.

Phylogenetic relationships in the stone fruit group of Prunus as revealed by restriction fragment analysis of chloroplast DNA.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.

Who is the mother of the potato? ? restriction endonuclease analysis of chloroplast DNA of cultivated potatoes

Chloroplast DNA from 44 lines of 16 wild and 7 cultivatedSolanum species were compared by restriction endonuclease analysis. Seven chloroplast genome types were identified among them by 5 restriction enzymes: Type A (S. tuberosum ssp.andigena andS. maglia); Type S (S. goniocalyx, S. phureja, S. stenotomum, S. ×chaucha and a line of ssp.andigena); Type C (S. acaule, S. bukasovii, S. canasense, S. multidissectum andS. ×juzepczukii); Type T (S. tuberosum ssp.tuberosum); Type W (other wild species); Type W' (S. chacoense f.gibberulosum) and Type W″ (S. tarijense). From this cytoplasmic identification, it was concluded thatS. goniocalyx, S. phureja, S. ×chaucha and ssp.andigena were derived fromS. stenotomum or its primitive type, which may have originally evolved itself fromS. canasense. The chloroplast genome of the European potato, however, was introduced from the Chilean potato, which might have been primarily constructed with the nuclear genome from ssp.andigena and with cytoplasm from other species. The cytoplasmic donor of the Chilean potato could not be determined.

Studies on the mode of separation of chloroplast genomes in parasexual hybrid calli. III Random separation of two types of chloroplast genomes in a hybrid callus.

In the parasexual hybrid callus between Nicotiana glauca and N. langsdorfii, the two parental types of chloroplasts separate during callus development. The system to investigate the mode of separation involves the hormone independent growth of the hybrid callus between N. glauca and N. langsdorfii as a selective marker for the parasexal hybrid and the isoelectrofocusing patterns of the large subunit (LS) of Fraction I protein as a chloroplast genetic marker. We improved this system by utilizing N. gossei as a chloroplast donor, because the isoelectrofocusing patterns of LS from N. gossei and that from N. langsdorfii can be distinguished clearly. The hormone independency which is suitable for selecting hybrids in the state of small calli was restored by transmitting the nuclear genome of N. glauca into N. gossei by means of sexual hybridization. Using the improved system, the mode of chloroplast separation was investigated in more detail. It was found that 84% of the cells in a two month old parasexual hybrid callus have only one or the other parental type of chloroplasts and 16% of the cells have two types.We calculated the rate of separation using the formulae based on the assumption that the two types of chloroplasts separate at random by cell divisions in the callus. The result obtained by the calculation turned out to be consistent to the experimental data obtained by the analysis of the two month old parasexual hybrid callus, showing that our assumption is reasonable.

Molecular basis of the genetic diversity of the cytoplasm in triticum and Aegilops. I. Diversity of the chloroplast genome and its lineage revealed by the restriction pattern of ct-DNAs.

Intact chloroplast DNAs were isolated from 34 self-fertile alloplasmic as well as from four euplasmic lines of wheats. The restriction patterns of these ct-DNAs were studied using four enzymes, Sal I, Pst I, Eco RI and Bam HI. Ct-DNAs from the 38 cytoplasms could be classified into three, four, four, and ten types, based on the Sal I, Pst I, Eco RI and Bam HI restriction patterns, respectively. In total, ten chloroplast genome types (Types 1 to 10) could be identified. The physical maps of ct-DNAs of common wheat (including emmer wheats and Ae. Longissima), Ae. Squarrosa (including its relatives, Ae. cylindrica and Ae. Ventricosa), and Ae. Uniaristata appeared to be about 0.2-0.5kbp smaller than that of most other species.Significantly the restriction patterns of ct-DNAs of Ae. caudata and an amphidiploid between Ae. caudata (_??_) and Ae, umbellulata (_??_) that had been produced 40 years ago were identical. No restriction pattern differences were found among three cultivars of common wheat (T. aestivum and T. spelta), two species of emmer wheat, two varieties of Ae. Biuncialis, three varieties of Ae. triaristata (one 4x and two 6x forms), and two strains of Ae. crassa (a 4x and 6x form). In many cases, a diploid and its polyploid relatives, which were previously assumed by Tsunewaki and his coworkers (ref. Tsunewaki 1980) to have received a nuclear genome and cytoplasm from the diploid, showed identical restriction patterns; one typical example is Ae. umbellulata (2x) and its relatives, Ae. triuncialis (4x), Ae. biuncialis (4x), Ae. columnaris (4x), and Ae. triaristata (4x and 6x). All these facts indicate a conservatism of chloroplast genomes during speciation.In all but two cases, each of the chloroplast genome types corresponded to one of the specific plasma types (or subtypes) established by Tsunewaki and his coworkers. One exceptional case is the chloroplast genome type 1 that included five distinctly different plasma types, A, D2, Sb, S1 and Sv Other exceptions are chloroplast genome types 7 and 8, both of which corresponded to the same plasma type, S. Since their differences appear on four restriction fragments, the plasma type corresponding to chloroplast genome type 7 (Ae. longissima, wild, and cultivated emmers and common wheat) has been renamed, B.Based on the number of restriction fragments differing between all pairs of the plasma types, a phylogenetic tree for 14 plasma types was constructed. Several characteristic genetic properties of the plasma types, such as large subunit types of Fraction I protein, induction of weakness, variegation, and male sterility, were localized in certain branches of the tree. However, none of them could be assigned to any specific change in the restriction fragments.Finally, the maternal lineages of most polyploid species were deduced from the restriction patterns of ct-DNAs: emmer and common wheats to Ae. longissima, 4x and 6x timopheevi wheats to Ae. aucheri, most 4x and 6x species of Polyeides section (Ae. triuncialis, Ae, biuncialis, Ae. columnaris, and 4x and 6x Ae. triaristata) to Ae. umbellulata, one strain of Ae. triuncialis to Ae. caudata, and Ae. cylindrica and Ae. ventricosa to Ae. squarrosa.