Subgeneric Clades Research Articles

The population of oomycetes in a recycled irrigation water system at a horticultural nursery in southern California

Recapture and recycling of irrigation water is often required to meet enormous water demands at horticultural nurseries. We tested four water types associated with a recycled irrigation system at a commercial container nursery in southern California for presence of oomycete plant pathogens from July 2015 to December 2017. These water types included: the main source of water originating from a reservoir, retention water from an on-site collection pond, irrigation water received by different growing areas within the nursery, and irrigation runoff captured in polyethylene sheet-lined runoff channels. The genera Phytophthora, Pythium, and Phytopythium together contributed more than 85% of the total oomycete population detected in the recycled irrigation system. The Phytophthora and Pythium genera were represented by member species from nine (1–4, 6–10) and eight (A, B, D-F, H-J) different sub-generic clades, respectively. Incoming water sourced from the reservoir was found to harbor known plant pathogens such as Phytophthora citricola-complex, P. capsici-cluster, P. tropicalis,P citrophthora-cluster, P. nemorosa-cluster, P. riparia, P. cryptogea-complex, P. parsiana-cluster, P. sp. nov. aff. kernoviae, Pythium dissotocum-complex, Py. oligandrum-cluster, Py. irregulare, and Phytopythium litorale. Runoff water showed the highest oomycete species richness and frequency of detection with both filtration and leaf baiting methods. In addition to plant pathogens, oomycete fish pathogens such as Aphanomyces laevis, Pythium chondricola-complex, Pythium flevoense-complex, and Saprolegnia diclina-complex were also detected in greater abundance in the recycled irrigation water. The oomycete species richness in the runoff water was correlated with several environmental parameters such as soil temperature. Greater oomycete richness in incoming water was associated with higher soil temperatures, whereas richness in runoff declines with increasing soil temperature, likely suggesting connections to weather-dependent nursery operations.

Mediterranean Ant Nest Beetles (Carabidae: Paussus): Out of Africa and Asia

Endemic to the Palaeotropic and southern Palaearctic regions, ant nest beetles (Carabidae: Paussus) are specialized predators that depend on ants for their survival. This obligate relationship has driven extreme morphological adaptations that obscured our understanding of Paussus species relationships and subgeneric clades for centuries. Molecular phylogenetics has revealed patterns of relationship with high levels of convergence, and as a general rule, areas of endemism are better predictors of monophyly than overall morphology. For example, the species rich fauna of Madagascar is the product of one dispersal event from Africa approximately 2.6 million years ago, after which Malagasy ant nest beetles undertook one of the fastest species radiations ever documented within animals. With their center of diversity in sub-Saharan Africa, the Paussus fauna of the Mediterranean is relatively depauperate with only seven species described from north of the Sahara. Pre-molecular subgeneric classifications, which were based on overall morphology, suggest that these seven species represent five species group lineages. Here, we use molecular sequence data from five genes and a taxon-sampling strategy aimed at investigating the biogeographic origins of five Mediterranean species. We find that the present-day Mediterranean fauna, unlike that of Madagascar, is the result of five separate dispersal events, four from the Afrotropical Region and one from the Indomalayan Region. Implications of associated host ant shifts are also explored.

Multiple origins of downy mildews and mito-nuclear discordance within the paraphyletic genus Phytophthora.

Phylogenetic relationships between thirteen species of downy mildew and 103 species of Phytophthora (plant-pathogenic oomycetes) were investigated with two nuclear and four mitochondrial loci, using several likelihood-based approaches. Three Phytophthora taxa and all downy mildew taxa were excluded from the previously recognized subgeneric clades of Phytophthora, though all were strongly supported within the paraphyletic genus. Downy mildews appear to be polyphyletic, with graminicolous downy mildews (GDM), brassicolous downy mildews (BDM) and downy mildews with colored conidia (DMCC) forming a clade with the previously unplaced Phytophthora taxon totara; downy mildews with pyriform haustoria (DMPH) were placed in their own clade with affinities to the obligate biotrophic P. cyperi. Results suggest the recognition of four additional clades within Phytophthora, but few relationships between clades could be resolved. Trees containing all twenty extant downy mildew genera were produced by adding partial coverage of seventeen additional downy mildew taxa; these trees supported the monophyly of the BDMs, DMCCs and DMPHs but suggested that the GDMs are paraphyletic in respect to the BDMs or polyphyletic. Incongruence between nuclear-only and mitochondrial-only trees suggests introgression may have occurred between several clades, particularly those containing biotrophs, questioning whether obligate biotrophic parasitism and other traits with polyphyletic distributions arose independently or were horizontally transferred. Phylogenetic approaches may be limited in their ability to resolve some of the complex relationships between the “subgeneric” clades of Phytophthora, which include twenty downy mildew genera and hundreds of species.

Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa 1998

ABSTRACT The family Rivulidae is the fourth most diverse clade of Neotropical fishes. Together with some genera of the related African family Nothobranchiidae, many rivulids exhibit a characteristic annual life cycle, with diapausing eggs and delayed embryonic development, which allows them to survive in the challenging seasonal ponds that they inhabit. Rivulidae also includes two species known as the only the self-fertilizing vertebrates and some species with internal fertilization. The first goal of this article is to review the systematics of the family considering phylogenetic relationships and synapomorphies of subfamilial clades, thus unifying information that is dispersed throughout the literature. From this revision, it is clear that phylogenetic relationships within Rivulidae are poorly resolved, especially in one of the large clades that compose it, the subfamily Rivulinae, where conflicting hypotheses of relationships of non-annual and annual genera are evident. The second goal of this work is to present an updated phylogenetic hypothesis (based on mitochondrial, nuclear, and morphological information) for one of the most speciose genus of Rivulidae, Austrolebias. Our results confirm the monophyly of the genus and of some subgeneric clades already diagnosed, but propose new relationships among them and their species composition, particularly in the subgenus Acrolebias.

The confounding effects of hybridization on phylogenetic estimation in the New Zealand cicada genus Kikihia

Phylogenetic studies of multiple independently inherited nuclear genes considered in combination with patterns of inheritance of organelle DNA have provided considerable insight into the history of species evolution. In particular, investigations of cicadas in the New Zealand genus Kikihia have identified interesting cases where mitochondrial DNA (mtDNA) crosses species boundaries in some species pairs but not others. Previous phylogenetic studies focusing on mtDNA largely corroborated Kikihia species groups identified by song, morphology and ecology with the exception of a unique South Island mitochondrial haplotype clade—the Westlandica group. This newly identified group consists of diverse taxa previously classified as belonging to three different sub-generic clades. We sequenced five nuclear loci from multiple individuals from every species of Kikihia to assess the nuclear gene concordance for this newly-identified mtDNA lineage. Bayes Factor analysis of the constrained phylogeny suggests some support for the mtDNA-based hypotheses, despite the fact that neither concatenation nor multiple species tree methods resolve the Westlandica group as monophyletic. The nuclear analyses suggest a geographic distinction between clearly defined monophyletic North Island clades and unresolved South Island clades. We suggest that more extreme habitat modification on South Island during the Pliocene and Pleistocene resulted in secondary contact and hybridization between species pairs and a series of mitochondrial capture events followed by subsequent lineage evolution.

The taxonomic placement of three fossil Fundulus species and the timing of divergence within the North American topminnows (Teleostei: Fundulidae).

The fossils species †Fundulus detillae, †F. lariversi, and †F. nevadensis from localities in the western United States are represented by well-preserved material with date estimations. We combined morphological data for these fossil taxa with morphological and DNA-sequence data to conduct a phylogenetic analysis and a tip-based divergence-time estimation for the family Fundulidae. The resultant phylogeny is largely concordant with the prior total-evidence phylogeny. The fossil species do not form a monophyletic group, and do not represent a discrete western radiation of Fundulus as previously proposed. The genus Fundulus diverged into subgeneric clades likely in the Eocene or Oligocene (mean age 34.6 mya, 53-23 mya), and all subgeneric and most species-group clades had evolved by the middle Miocene. †Fundulus lariversi is a member of subgenus Fundulus in which all extant species are found only in eastern North America, demonstrating that fundulids had a complicated biogeographic history. We confirmed †Fundulus detillae as a member of the subgenus Plancterus. †F. nevadensis is not classified in a subgenus but likely is related to the subgenera Plancterus and Wileyichthys.

Symbiodinium (Dinophyceae) community patterns in invertebrate hosts from inshore marginal reefs of the southern Great Barrier Reef, Australia.

The broad range in physiological variation displayed by Symbiodinium spp. has proven imperative during periods of environmental change and contribute to the survival of their coral host. Characterizing how host and Symbiodinium community assemblages differ across environmentally distinct habitats provides useful information to predict how corals will respond to major environmental change. Despite the extensive characterizations of Symbiodinium diversity found amongst reef cnidarians on the Great Barrier Reef (GBR) substantial biogeographic gaps exist, especially across inshore habitats. Here, we investigate Symbiodinium community patterns in invertebrates from inshore and mid-shelf reefs on the southern GBR, Australia. Dominant Symbiodinium types were characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Twenty one genetically distinct Symbiodinium types including four novel types were identified from 321 reef-invertebrate samples comprising three sub-generic clades (A, C, and D). A range of host genera harbored C22a, which is normally rare or absent from inshore or low latitude reefs in the GBR. Multivariate analysis showed that host identity and sea surface temperature best explained the variation in symbiont communities across sites. Patterns of changes in Symbiodinium community assemblage over small geographic distances (100s of kilometers or less) indicate the likelihood that shifts in Symbiodinium distributions and associated host populations, may occur in response to future climate change impacting the GBR.

Symbiodinium (Dinophyceae) diversity in reef‐invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef

Despite extensive work on the genetic diversity of reef invertebrate-dinoflagellate symbioses on the Great Barrier Reef (GBR; Australia), large information gaps exist from northern and inshore regions. Therefore, a broad survey was done comparing the community of inshore, mid-shelf and outer reefs at the latitude of Lizard Island. Symbiodinium (Freudenthal) diversity was characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Thirty-nine distinct Symbiodinium types were identified from four subgeneric clades (B, C, D, and G). Several Symbiodinium types originally characterized from the Indian Ocean were discovered as well as eight novel types (C1kk, C1LL, C3nn, C26b, C161a, C162, C165, C166). Multivariate analyses on the Symbiodinium species diversity data showed a strong link with host identity, consistent with previous findings. Of the four environmental variables tested, mean austral winter sea surface temperature (SST) influenced Symbiodinium distribution across shelves most significantly. A similar result was found when the analysis was performed on Symbiodinium diversity data of genera with an open symbiont transmission mode separately with chl a and PAR explaining additional variation. This study underscores the importance of SST and water quality related variables as factors driving Symbiodinium distribution on cross-shelf scales. Furthermore, this study expands our knowledge on Symbiodinium species diversity, ecological partitioning (including host-specificity) and geographic ranges across the GBR. The accelerating rate of environmental change experienced by coral reef ecosystems emphasizes the need to comprehend the full complexity of cnidarian symbioses, including the biotic and abiotic factors that shape their current distributions.

Phylogenetic Relationships in the North American GenusPseudemys(Emydidae) Inferred from Two Mitochondrial Genes

Abstract Pseudemys turtles are an important component of southeastern North American aquatic ecosystems, but the relationships within the genus are poorly understood. Convergent morphology and apparent hybridization have complicated the identification of species boundaries and have resulted in numerous conflicting taxonomic treatments. We used mitochondrial DNA sequence data from the control region and cytochrome-b gene to address 1 ) the monophyly of currently recognized subgeneric clades (the cooters and the red-bellies), and 2) relationships within these two groups. A total of 91 specimens representing 8 Pseudemys and 3 outgroup taxa were sampled, and 36 distinct haplotypes were recovered. Pseudemys forms a well-supported monophyletic group, but relationships among species were not well resolved, such that support for the two subgeneric groupings was lacking. Furthermore, most taxa do not appear to be monophyletic, with the exception of P. gorzugi (Rio Grande Cooter) and P. texana (Texas Cooter), sugge...

Phylogenetics and the evolution of major structural characters in the giant genus Euphorbia L. (Euphorbiaceae)

Euphorbia is among the largest genera of angiosperms, with about 2000 species that are renowned for their remarkably diverse growth forms. To clarify phylogenetic relationships in the genus, we used maximum likelihood, Bayesian, and parsimony analyses of DNA sequence data from 10 markers representing all three plant genomes, averaging more than 16kbp for each accession. Taxon sampling included 176 representatives from Euphorbioideae (including 161 of Euphorbia). Analyses of these data robustly resolve a backbone topology of four major, subgeneric clades—Esula, Rhizanthium, Euphorbia, and Chamaesyce—that are successively sister lineages. Ancestral state reconstructions of six reproductive and growth form characters indicate that the earliest Euphorbia species were likely woody, non-succulent plants with helically arranged leaves and 5-glanded cyathia in terminal inflorescences. The highly modified growth forms and reproductive features in Euphorbia have independent origins within the subgeneric clades. Examples of extreme parallelism in trait evolution include at least 14 origins of xeromorphic growth forms and at least 13 origins of seed caruncles. The evolution of growth form and inflorescence position are significantly correlated, and a pathway of evolutionary transitions is supported that has implications for the evolution of Euphorbia xerophytes of large stature. Such xerophytes total more than 400 species and are dominants of vegetation types throughout much of arid Africa and Madagascar.

Systematic palynology in Ebenaceae with focus on Ebenoideae: Morphological diversity and character evolution

This study examines the diversity and character transformations in pollen and orbicule morphology of Ebenaceae, with a focus on subfamily Ebenoideae (ca. 600 sp.). 62 specimens comprising all three genera of Ebenoideae ( Diospyos, Euclea, Royena), were studied using LM and SEM. Bayesian phylogenetic analysis was performed on molecular sequence data to establish an evolutionary hypothesis that was then used as an evolutionary framework to identify synapomorphies and trace evolutionary trends of palynological data with Bayesian posterior mapping and principle component analyses (PCA). Ebenoideae pollen is generally shed as monads (permanent tetrads in two species), medium-sized, prolate-spheroidal to subprolate and tricolporate. A substantial amount of variation is found in pollen size, equatorial outline (lobate, subtriangular, circular and hexagonal) and sexine ornamentation type ((micro)rugulate, striate, granulate and gemmate). Moreover, orbicules were present on the inner locule wall in all specimens examined. Their abundance, degree of fusion with tapetal membrane and aggregation vary considerably. We can conclude that Ebenaceae pollen is more heterogeneous than previously assumed. We traced palynological synapomorphies for groups at different taxonomic levels: subfamily level (pollen size, pollen wall stratification and aperture morphology), generic level (size, equatorial outline and sexine ornamentation types) and subgeneric clades (size, ectocolpus morphology, equatorial outline and sexine ornamentation subtypes) respectively. The granular infratectum and the unique sculpturing pattern on the orbicule walls are the most discriminating pollen features for subfamily Ebenoideae.

Gene-Specific Signatures of Elevated Non-Synonymous Substitution Rates Correlate Poorly across the Plasmodium Genus

BackgroundComparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages.Principal FindingsHere, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes.SignificanceThe inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi.

Molecular phylogenetics of the butterflyfishes (Chaetodontidae): Taxonomy and biogeography of a global coral reef fish family

Marine butterflyfishes (10 genera, 114 species) are conspicuously beautiful and abundant animals found on coral reefs worldwide, and are well studied due to their ecological importance and commercial value. Several phylogenies based on morphological and molecular data exist, yet a well-supported molecular phylogeny at the species level for a wide range of taxa remains to be resolved. Here we present a molecular phylogeny of the butterflyfishes, including representatives of all genera (except Parachaetodon) and at least one representative of all commonly cited subgenera of Chaetodon (except Roa sensu Blum, 1988). Genetic data were collected for 71 ingroup and 13 outgroup taxa, using two nuclear and three mitochondrial genes that total 3332 nucleotides. Bayesian inference, parsimony, and maximum likelihood methods produced a well-supported phylogeny with strong support for a monophyletic Chaetodontidae. The Chaetodon subgenera Exornator and Chaetodon were found to be polyphyletic, and the genus Amphichaetodon was not the basal sister group to the rest of the family as had been previously proposed. Molecular phylogenetic analysis of data from 5 genes resolved some clades in agreement with previous phylogenetic studies, however the topology of relationships among major butterflyfish groups differed significantly from previous hypotheses. The analysis recovered a clade containing Amphichaetodon, Coradion, Chelmonops, Chelmon, Forcipiger, Hemitaurichthys, Johnrandallia, and Heniochus. Prognathodes was resolved as the sister to all Chaetodon, as in previous hypotheses, although the topology of subgeneric clades differed significantly from hypotheses based on morphology. We use the species-level phylogeny for the butterflyfishes to resolve long-standing questions regarding the use of subgenera in Chaetodon, to reconstruct molecular rates and estimated dates of diversification of major butterflyfish clades, and to examine global biogeographic patterns.

Phylogenetic systematics of the Gondwanan nephilid spider lineage Clitaetrinae (Araneae, Nephilidae)

This study revises the taxonomy, biology, phylogeny, and biogeography of the basal‐most nephilid spider lineage, the Clitaetrinae, with the least known nephilid genus Clitaetra. The five previously known species are redescribed: Clitaetra clathrata Simon from western Africa, C. simoni Benoit from central Africa, C. episinoides Simon from the Comoro Islands and Mayotte, C. perroti Simon from Madagascar, and C. thisbe Simon from Sri Lanka with first descriptions of the males of C. clathrata and C. perroti. Additionally, C. irenae sp. nov. is described in both sexes from southern Africa. Clitaetra biology, so far largely unknown, is presented here based on observations of C. irenae in South Africa, and clitaetrine anatomy is summarized to assess phylogenetic homologies. A species‐level phylogenetic analysis of 32 taxa scored for 197 morphological and behavioural characters results in eight most parsimonious cladograms and places Clitaetra as sister to the clade (Herennia+ (Nephilengys+Nephila)). Thus, the orb‐weaving spider family Nephilidae Simon contains the (sub)tropical genera Nephila, Nephilengys, Herennia, and Clitaetra, but not Deliochus or Phonognatha. Contra recent cladistic treatments, the nephilines are not tetragnathids, but the sister group to the newly proposed clade, Nephilidae, is ambiguous. The three species clades (subgenera) within Clitaetra show a seemingly old Gondwanan biogeographic pattern: Afroetra subgen. nov., with the three mainland African species, is sister to Clitaetra with the two Indian Ocean island species. Indoetra subgen. nov. contains the unstudied species from Sri Lanka, C. thisbe. Future understanding of the morphology and biology of C. thisbe is important for the polarization of many nephilid features. Vicariance would estimate the clitaetrine subgeneric clades and basal nephilid lineages to be at least 160 Myr old and of Gondwanan origin.

Molecular Evidence for Host–Symbiont Specificity in Soritid Foraminifera

Symbiosis between the dinoflagellate genus Symbiodinium and various invertebrates and protists is an ubiquitous phenomenon in shallow tropical and subtropical waters. Molecular studies undertaken on cnidarian symbionts revealed the presence of several distinctive lineages or subgeneric clades of Symbiodinium whose taxonomic level provides limited information about the specificity between invertebrate hosts and their symbionts. This contrasts with the finding of several Symbiodinium clades being present almost exclusively in foraminifera and belonging to the subfamily Soritinae. To test whether such specificity also exists at a lower taxonomic level within Soritinae, we obtained the SSU rDNA sequences from 159 soritid individuals collected in nine localities worldwide and representing all known morphospecies of this subfamily. For each individual, the symbionts were determined either by sequencing or by RFLP analysis. We distinguished 22 phylotypes of Soritinae in relation with a number of symbiont "groups" corresponding to 3 clades and 5 subclades of Symbiodinium. Among the 22 soritid phylotypes, 14 show strict symbiont specificity and only one was found to be a host for more than two "groups" of Symbiodinium. It is suggested that the strong host-symbiont specificity observed in Soritinae is a combined effect of a selective recognition mechanism, vertical transmission of symbionts, and biogeographical isolation.

On the success of a swindle: pollination by deception in orchids

A standing enigma in pollination ecology is the evolution of pollinator attraction without offering reward in about one third of all orchid species. Here I review concepts of pollination by deception, and in particular recent findings in the pollination syndromes of food deception and sexual deception in orchids. Deceptive orchids mimic floral signals of rewarding plants (food deception) or mating signals of receptive females (sexual deception) to attract pollen vectors. In some food deceptive orchids, similarities in the spectral reflectance visible to the pollinator in a model plant and its mimic, and increased reproductive success of the mimic in the presence of the model have been demonstrated. Other species do not mimic specific model plants but attract pollinators with general attractive floral signals. In sexually deceptive orchids, floral odor is the key trait for pollinator attraction, and behaviorally active compounds in the orchids are identical to the sex pheromone of the pollinator species. Deceptive orchids often show high variability in floral signals, which may be maintained by negative frequency-dependent selection, since pollinators can learn and subsequently avoid common deceptive morphs more quickly than rare ones. The evolution of obligate deception in orchids seems paradoxical in the light of the typically lower fruit set than in rewarding species. Pollination by deception, however, can reduce self-pollination and encourage pollen flow over longer distances, thus promoting outbreeding. Although some food deceptive orchids are isolated through postzygotic reproductive barriers, sexually deceptive orchids lack post-mating barriers and species isolation is achieved via specific pollinator attraction. Recent population genetic and phylogenetic investigations suggest gene-flow within subgeneric clades, but pollinator-mediated selection may maintain species-specific floral traits.

Specific pollinator attraction and the diversification of sexually deceptive Chiloglottis (Orchidaceae)

Evidence indicates that sexually deceptive Chiloglottis R.Br. (Orchidaceae) taxa specifically attract their thynnine wasp (Tiphiidae) pollinators through the floral odour mimicry of female wasp sex pheromones. We use amplified fragment length polymorphisms (AFLPs) to reconstruct the species-level phylogeny of Chiloglottis, make a preliminary evaluation of genetic distinctions between species, and compare the historical association among orchids and their pollinators using wasp sequence data from a previous study. AFLPs show large differences between three sub-generic clades relative to that found among species within each clade. Interspecific genetic barriers are indicated by AFLP discontinuities among species unlike in previously reported DNA sequence data. However, such barriers are demonstrated clearly in only one of the two pairs of sympatric species sampled more intensively. We interpret these patterns as indicating either (i) a rapid and recent radiation of species within each clade following histories of stasis or extinction, or (ii) alternating cycles of divergence and gene flow acting to homogenize genetic differences among species within each of the three clades.

Evolution of Mitochondrial and Ribosomal Gene Sequences in Anophelinae (Diptera: Culicidae): Implications for Phylogeny Reconstruction

In this study, two mitochondrial genes, cyt b and ND5, and the D2 expansion segment of the 28S nuclear ribosomal gene were used to reconstruct a phylogeny of the mosquito subfamily Anophelinae. The ingroup consisted of all three genera of Anophelinae and five of six subgenera of Anopheles. Six genera of Culicinae were used as the outgroup. Extreme conservation at the protein level coupled with rapid saturation of synonymous positions probably accounted for the lack of meaningful phylogenetic signal in the cyt b gene. In contrast, abundant variation at all codon positions of the ND5 gene allowed recovery of the basal and most of the recent relationships. Phylogenetic analysis of D2 produced results consistent with those of ND5. Combined analysis indicated well-supported monophyletic Anophelinae (with Chagasia basal), Anopheles + Bironella, and subgeneric clades within the genus Anopheles. Moreover, subgenera Nyssorhynchus and Kerteszia were supported as a monophyletic lineage. The Kishino–Hasegawa test could not reject the monophyly of Anopheles, whereas the recently proposed hypothesis of close affinity of Bironella to the subgenus Anopheles was rejected by the analyses of ND5 and combined data sets. The lack of resolution of Bironella and Anopheles clades, or basal relationships among subgeneric clades within Anopheles, suggests their rapid diversification. Recovery of relationships consistent with morphology and previous molecular studies provides evidence of substantial phylogenetic signal in D2 and ND5 genes at levels of divergence from closely related species to subfamily in mosquitoes.