Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Phylogeny and evolution of mycophagy in the Zygothrica genus group (Diptera: Drosophilidae).

Despite numerous phylogenetic studies on the family Drosophilidae, relationships among some important lineages are still poorly resolved. An example is the equivocal position of the Zygothrica genus group that is mostly comprised of the mycophagous genera Hirtodrosophila, Mycodrosophila, Paramycodrosophila, and Zygothrica. To fill this gap, we conducted a phylogenetic analysis by assembling a dataset of 24 genes from 92 species, including 42 species of the Zygothrica genus group mainly from the Palearctic and Oriental regions. The resulting tree shows that the Zygothrica genus group is monophyletic and places it as the sister to the genus Dichaetophora, and the clade Zygothrica genus group + Dichaetophora is sister to the Siphlodora + Idiomyia/Scaptomyza clade. Within the Zygothrica genus group, the genera Mycodrosophila and Paramycodrosophila are both recognized as monophyletic, while neither the genus Zygothrica nor Hirtodrosophila is monophyletic. We also used this phylogenetic tree to investigate the evolution of mycophagy by reconstructing ancestral food habit in the Drosophilidae. We found that fungus-feeding habit has been gained independently in two lineages. The most recent common ancestor (MRCA) of the subgenus Drosophila was estimated to have acquired mycophagy by expanding its ancestral feeding niche on fermenting fruits to decayed fungi, while the MRCA of the Zygothrica genus group shifted its niche from fruits to fungi as a specialist probably preferring fresh fruiting bodies.

Evolution and diversity of the courtship repertoire in the Drosophila montium species group (Diptera: Drosophilidae).

Changes in elements of courtship behaviour can influence sexual isolation between species. Large-scale analyses of changes, including loss and gain of courtship elements, across a relatively complete phylogenetic group are rare but needed to understand the significance of such changes, for example whether the gain and loss of courtship elements are essentially arbitrary or equally reversible. In most species of Drosophila, courtship, including singing, mainly occurs before mounting as premounting courtship. The Drosophila montium species group is unusual because loss of premounting courtship and gain of post-mounting one has been detected in this group. Here, we provide an extensive analysis on the courtship repertoire and songs of 42 species in this group. Synchronously captured video and audio recordings were analysed to describe courtship patterns and male courtship songs, and changes were analysed in a phylogenetic context. Ancestral state reconstruction suggests that a gain of post-mounting courtship singing at the ancestor of this species group has been accompanied by a concurrent decrease in the incidence of premounting courtship singing and has led to subsequent further decrease and eventually complete loss of premounting courtship song in several lineages. Alongside this evolutionary trend towards post-mounting courtship, sine song and a special type of "high pulse repetition song" have become more widely used for courtship during species diversification in the montium group. It is likely that the elaboration of post-mounting courtship behaviours is associated with changes in the relative importance of pre- and post-mounting components of mating systems, such as sperm competition or cryptic female choice.

Molecular Phylogeny of Cypridoid Freshwater Ostracods (Crustacea: Ostracoda), Inferred from 18S and 28S rDNA Sequences.

With the aim of exploring phylogenetic relationships within Cypridoidea, the most species-rich superfamily among the podocopidan ostracods, we sequenced nearly the entire 18S rRNA gene (18S) and part of the 28S rRNA gene (28S) for 22 species in the order Podocopida, with representatives from all the major cypridoid families. We conducted phylogenetic analyses using the methods of maximum likelihood, minimum evolution, and Bayesian analysis. Our analyses showed monophyly for Cyprididae, one of the four families currently recognized in Cypridoidea. Candonidae turned out to be paraphyletic, and included three clades corresponding to the subfamilies Candoninae, Paracypridinae, and Cyclocypridinae. We propose restricting the name Candonidae s. str. to comprise what is now Candoninae, and raising Paracypridinae and Cyclocyprininae to family rank within the superfamily Cypridoidea.

Introgression and Habitat Segregation in a Pair of Ladybird Beetle Species in the Genus Propylea (Coccinellidae, Coccinellinae) in Northern Japan.

The ladybird beetles Propylea quatuordecimpunctata and P. japonica have largely overlapping distributions in northern Japan, and in the laboratory produce fertile hybrids. In this study, we surveyed the distribution and morphological differentiation of these species and the hybrids in natural populations, with a focus on western Hokkaido, northern Japan. Phenotypic analyses were conducted for 987 individuals collected at 90 localities. In addition, the nuclear internal transcribed spacer-II (ITS2) region (549 bp) and part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (700 bp) were sequenced for 620 individuals from 53 localities. Analyses of both phenotypic and genotypic features discriminated two distinct entities assignable to P. quatuordecimpunctata and P. japonica. However, individuals with intermediate phenotypes and/or genotypes also occurred extensively, indicating natural hybridization. Putative P. quatuordecimpunctata individuals were collected across a wide range of altitudes (30-600 m), whereas those of P. japonica were found mostly lower than 300 m alt. In addition, P. quatuordecimpunctata was dominant in semi-open habitats shaded by canopy foliage, whereas P. japonica was frequent in more open habitats. The perceived altitudinal difference in the distributions may thus in part be a consequence of this different habitat preference, as open habitats are more common at lower altitudes in the study area.

Phylogeny of epilachna, henosepilachna, and some minor genera of phytophagous ladybird beetles (coleoptera: coccinellidae: coccinellinae: epilachnini), with an analysis of ancestral biogeography and host-plant utilization.

Ladybird beetles in the tribe Epilachnini include notorious crop pests and model species studied intensively in various fields of evolutionary biology. From a combined dataset of mitochondrial (ND2) and nuclear (28S) DNA sequences, we reconstructed the phylogeny of 46 species of Epilachnini from Asia, Africa, America, and the Australian region: 16 species in Epilachna, 24 species in Henosepilachna, and one species each in Adira, Afidenta, Afidentula, Afissula, Chnootriba, and Epiverta. In our phylogenetic trees, both Epilachna and Henosepilachna were reciprocally polyphyletic. Asian Epilachna species were monophyletic, except for the inclusion of Afissula sp. Asian and Australian Henosepilachna species likewise formed a monophyletic group, excluding H. boisduvali. African Epilachna and Henosepilachna species did not group with their respective Asian and American congeners, but were paraphyletic to other clades (Epilachna species) or formed a separate monophyletic group (Henosepilachna species) together with Chnootriba similis. The American Epilachna species were monophyletic and formed a clade with American Adira clarkii and Asian Afidentula manderstjernae bielawskii; this clade was the sister group to Asian and Australian Henosepilachna, but was distant from Asian Epilachna. Chnootriba was embedded in the African Henosepilachna clade, and Afissula in the Asian Epilachna clade. Epiverta, which is morphologically unique, was the sister group to Asian Epilachna, although with weak support. From reconstructions of biogeographical distribution and host-plant utilization at ancestral nodes, we inferred an African origin for the common ancestor of the species studied, and found the frequency of host shifts to differ greatly between the two major lineages of Epilachnini examined.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Phylogenetic relationships between Sophophora and Lordiphosa, with proposition of a hypothesis on the vicariant divergences of tropical lineages between the Old and New Worlds in the family Drosophilidae.

Despite many studies on the phylogeny of the subgenus Sophophora, its monophyly has not been established, especially in relation to its putative relative, the genus Lordiphosa. We analyzed their phylogenetic relationships using DNA sequence data of two mitochondrial genes (ND2 and COII) and two nuclear genes (Adh and 28SrRNA). In constructing phylogenetic trees, we accounted for the problem of among-taxa nucleotide compositional heterogeneity, and took a sequence-partitioning approach to allow multiple substitution models for nucleotide sequences that have evolved under different evolutionary processes, particularly developing a novel, sequence-partitioning procedure for Neighbor Joining (NJ) tree construction. Trees constructed by different methods showed an almost identical and strongly supported topology in which Sophophora was paraphyletic: Lordiphosa was placed as the sister to the Neotropical Sophophora consisting of the saltans and willistoni groups, and Sophophora was divided into the clade of Lordiphosa+Neotropical Sophophora and the clade of the obscura+melanogaster groups. Based on the estimated time, 45.9 Mya, of divergence between the Old World Lordiphosa and the Neotropical Sophophora and evidence from paleontology, paleo-geography and -climatology, we propose a hypothesis that this vicariant divergence should have occurred when the North Atlantic Land Bridge between Europe and North America broke in the middle Eocene Epoch.

Molecular systematics of Tanaidacea (Crustacea: Peracarida) based on 18S sequence data, with an amendment of suborder/superfamily-level classification.

Phylogenetic relationships within Tanaidacea were analyzed based on sequence data for the 18S rRNA gene. Our results strongly supported a monophyletic group composed of Neotanaidae, Tanaoidea, and Paratanaoidea, with the first two taxa forming a clade. These results contradict three previously suggested hypotheses of relationships. Based on the molecular results, and considering morphological similarities/differences between Neotanaidomorpha and Tanaidomorpha, we demoted Suborder Neotanaidomorpha to Superfamily Neotanaoidea within Tanaidomorpha; with this change, the classification of extant tanaidaceans becomes a two-suborder, four-superfamily system. This revision required revision of the diagnoses for Tanaidomorpha and its three super-families. The results for Apseudomorpha were ambiguous: this taxon was monophyletic in the maximum likelihood and Bayesian analyses, but paraphyletic in the maximum parsimony and minimum evolution analyses.

Evolution of a neuromuscular sexual dimorphism in the Drosophila montium species group.

While epigamic traits likely evolve via sexual selection, the mechanism whereby internal sexual dimorphism arises remains less well understood. Seeking clues as to how the internal sexual dimorphism evolved, we compared the abdominal musculature of 41 Drosophila montium group species, to determine whether any of these species carry a male-specific muscle of Lawrence (MOL). Our quantitative analysis revealed that the size of a sexually dimorphic MOL analog found in 19 montium group species varied widely from species to species, suggesting the gradual evolution of this sexually dimorphic neuromuscular trait. We attempted the ancestral state reconstitution for the presence or absence of the neuromuscular sexual dimorphism in the A5 segment; the neuromuscular sexual dimorphism existed in an old ancestor of the montium group, which was lost in some of the most recent common ancestors of derived lineages, and subsequently some species regained it. This loss-and-gain history was not shared by evolutionary changes in the courtship song pattern, even though both traits were commonly regulated by the master regulator male-determinant protein FruM. It is envisaged that different sets of FruM target genes may serve for shaping the song and MOL characteristics, respectively, and, as a consequence, each phenotypic trait underwent a distinct evolutionary path.

DrosoPhyla: Resources for Drosophilid Phylogeny and Systematics.

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Effects of straits as dispersal barriers for the flightless roving carrion beetle, Silpha perforata (Coleoptera, Silphidae, Silphinae).

Population genetic analyses were conducted for the flightless beetle Silpha perforata in northern Japan to evaluate effects of straits as dispersal barriers. In all, 19 populations on the Hokkaido mainland and one population on each of seven adjacent islands were included in the study. Based on partial sequences (853 bp) of the mitochondrial ND2 gene from 365 individuals, we calculated haplotype diversity (h), nucleotide diversity (pi), Tajima's D, and Fu's F(s) statistics for each population. The genetic distance d(A) and fixation index F(ST) were then estimated between populations, excluding two mainland and two island populations with small sample sizes. The results showed that four island populations (Rebun, Rishiri, Yagishiri, and Teuri) were genetically diverged and isolated from mainland populations, including the closest ones, consistent with the assumption that the straits separating these islands from the mainland have functioned as strong barriers against beetle dispersal. However, some factors other than sea barriers may have resulted in the observed divergence of these island populations, since the straits might be too young to permit such divergence (all are estimated to have been finally established approximately 11,000 to 13,000 years BP). On the other hand, the population on Okushiri Island showed little genetic divergence from the nearest mainland population, suggesting current overseas dispersal across the strait (estimated to have been formed more than 200,000 years BP). The present study thus demonstrated that the effects of straits on dispersal can be diverse, even for species of terrestrial invertebrates with limited dispersal ability.

Taxonomy and evolution of asymmetric male genitalia in the subgenus Ashima Chen (Diptera: Drosophilidae: Phortica Schiner), with descriptions of seven new species.

The taxonomy of the subgenus Ashima of the genus Phortica is revised. A cladistic analysis of 66 morphological characters is conducted, covering 35 species (28 known and 7 new species: Phortica efragmentata sp. nov., P. andreagigoni sp. nov., P. watabei sp. nov., P. halimunensis sp. nov., P. akutsui sp. nov., P. kerinciensis sp. nov., and P. takehiroi sp. nov.) which correspond to 71.4% of 49 total spp. of Ashima. The resulting cladogram shows that the studied species are separated into several clades/subclades/cluster each highly supported with specific synapomorphies. Those clades/subclades/cluster are newly defined as the following species groups, subgroups or complex: the afoliolata, foliiseta, nigrifoliiseta and angulata species groups; the foliiseta, tanabei, nigrifoliiseta and glabra species subgroups; and the foliiseta species complex. The subgenus Ashima is peculiar in having the asymmetric male genitalia as the ground plan and showing the antisymmetry (i.e. intraspecific mirror-image variation) in some species but the directional asymmetry (i.e. side-fixed asymmetry) in others. The evolution of genital asymmetry in this subgenus is estimated by mapping the states (symmetry, directional asymmetry and antisymmetry) of bilateral structures of male genitalia on the cladogram. This ancestral state reconstruction estimates that the directional asymmetry of male genitalia has evolved at the ancestor of this subgenus and then changed to the antisymmetric state independently in two lineages, the angulata + nigrifoliiseta species groups and the foliiseta species complex. In this study, a standardized terminology recently proposed for the male terminalia of Drosophila melanogaster is extendedly adopted to describe the morphology of male terminalia in the subgenus Ashima, one group of the subfamily Steganinae.

Phylogeny of the Drosophila immigrans species group (Diptera: Drosophilidae) based on Adh and Gpdh sequences.

The immigrans species group in the Drosophilinae is one of the representative species groups of Drosophila in East Asia. Although this group constitutes a significant part of the drosophilid fauna in the Old World, only a few species have been analyzed in previous molecular phylogenetic studies. To study the phylogeny of the immigrans group, we analyzed the nucleotide sequences of two nuclear genes, alcohol dehydrogenase (Adh) and glycerol-3-phosphate dehydrogenase (Gpdh), for 36 drosophilid species, including 12 species of the immigrans group. In the resultant phylogenetic trees, 10 species of the immigrans group (D. immigrans, D. formosana, D. ruberrima, D. albomicans, D. nasuta, D. neonasuta, D. pallidifrons, D. hypocausta, D. neohypocausta, D. siamana) consistently formed a clade (the immigrans group proper), although the phylogeny within this clade did not exactly correspond to the classification of species subgroups. However, D. annulipes and D. quadrilineata, both of which belong to the quadrilineata subgroup of the immigrans group, were not included in the immigrans group proper. Furthermore, we obtained the unexpected result that D. annulipes was included in a clade comprising Scaptomyza and Hawaiian Drosophila, together with D. maculinotata of the funebris group, although the phylogenetic relationships within this clade remain uncertain and need to be substantiated with further studies. Thus, according to the present study, the immigrans group is polyphyletic.

Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): Evolutionary History of Faunal Divergence between the Old and the New Worlds.

The current subgenus Drosophila (the traditional immigrans-tripunctata radiation) includes major elements of temperate drosophilid faunas in the northern hemisphere. Despite previous molecular phylogenetic analyses, the phylogeny of the subgenus Drosophila has not fully been resolved: the resulting trees have more or less varied in topology. One possible factor for such ambiguous results is taxon-sampling that has been biased towards New World species in previous studies. In this study, taxon sampling was balanced between Old and New World species, and phylogenetic relationships among 45 ingroup species selected from ten core species groups of the subgenus Drosophila were analyzed using nucleotide sequences of three nuclear and two mitochondrial genes. Based on the resulting phylogenetic tree, ancestral distributions and divergence times were estimated for each clade to test Throckmorton's hypothesis that there was a primary, early-Oligocene disjunction of tropical faunas and a subsequent mid-Miocene disjunction of temperate faunas between the Old and the New Worlds that occurred in parallel in separate lineages of the Drosophilidae. Our results substantially support Throckmorton's hypothesis of ancestral migrations via the Bering Land Bridge mainly from the Old to the New World, and subsequent vicariant divergence of descendants between the two Worlds occurred in parallel among different lineages of the subgenus Drosophila. However, our results also indicate that these events took place multiple times over a wider time range than Throckmorton proposed, from the late Oligocene to the Pliocene.

Genetic features of Khoton Mongolians revealed by SNP analysis of the X chromosome.

The Khoton Mongolian population is a small and relatively isolated ethnic group residing predominantly in the northwestern part of Mongolia. A recent genetic study of the Y chromosome revealed that the major Mongolian ethnic groups have a relatively close genetic affinity to populations in the northern part of East Asia, while the Khoton population reflected an apparent genetic differentiation from the other Mongolian populations. To further investigate the genetic features of the Khoton and the other Mongolian populations, we analyzed the single nucleotide polymorphisms (SNPs) in the Xq13.3 region, which is thought to have an extremely low level of recombination in the human X chromosome. We found that the frequency distribution of Xq13.3 haplotypes in the Khoton population was substantially different from those in three other Mongolian populations (Khalkh, Uriankhai, and Zakhchin). The same relationship was also revealed by the results from the population tree and principal-component (PC) analysis based on the allele frequencies. These results are largely consistent with the hypothesis that the Khoton population descended from a nomadic tribe of Turkish origin, which has been supported by previous anthropological, historical, and Y-chromosome studies. However, the population structure analysis produced an additional finding, namely, that the Khoton population is likely to be an admixed population.

Genetic features of Mongolian ethnic groups revealed by Y-chromosomal analysis.

About 20 ethnic groups reside in Mongolia. On the basis of genetic and anthropological studies, it is believed that Mongolians have played a pivotal role in the peopling of Central and East Asia. However, the genetic relationships among these ethnic groups have remained obscure, as have their detailed relationships with adjacent populations. We analyzed 16 binary and 17 STR polymorphisms of human Y chromosome in 669 individuals from nine populations, including four indigenous ethnic groups in Mongolia (Khalkh, Uriankhai, Zakhchin, and Khoton). Among these four Mongolian populations, the Khalkh, Uriankhai, and Zakhchin populations showed relatively close genetic affinities to each other and to Siberian populations, while the Khoton population showed a closer relationship to Central Asian populations than to even the other Mongolian populations. These findings suggest that the major Mongolian ethnic groups have a close genetic affinity to populations in northern East Asia, although the genetic link between Mongolia and Central Asia is not negligible.

Phylogeny and evolution of butterflies of the genus Parnassius: inferences from mitochondrial 16S and ND1 sequences.

Phylogenetic relationships among species of the genus Parnassius and its related taxa were analyzed by comparing nucleotide sequences of mitochondrial 16S ribosomal RNA (504 sites) and NADH-dehydrogenase subunit 1 (469 sites). In the phylogenetic trees, Parnassius was found to be most closely related to Hypermnestra helios, whereas Archon apollinus, which has been classified in the tribe Parnassiini together with Parnassius and Hypermnestra, was more closely related to members of the tribe Zerynthiini. Within the Parnassius clade, six major clades corresponding to species groups were well supported, although the phylogenetic relationships among them were not clear. Although the results of the present study were in agreement with those of a previous phylogenetic study based on mitochondrial NADH-dehydrogenase subunit 5 sequences, our study strongly supported a close relationship between Parnassius and Hypermnestra, which was not well supported in the previous study.

Molecular systematics and evolution of the "Apollo" butterflies of the genus Parnassius (Lepidoptera: Papilionidae) based on mitochondrial DNA sequence data.

Sequences of 777 bp of mtDNA-ND5 locus were determined in order to shed light on the molecular systematics and evolution of the "Apollo" butterflies. Examined were nearly all of about 50 species of the genus Parnassius, together with seven species of the allied genera in the subfamily Parnassiinae (Papilionidae). The NJ and the MP phylogenetic trees show that the "Apollos" constitute a monophyletic group, comprising a number of cluster groups probably reflecting a relatively rapid radiation in evolution. The clusters of species-groups denoted I-VIII correspond to those species-groups recognized on the basis of morphological characters. Our findings will also help understand the biological relationships among several species or subspecies on which the classical taxonomy is in dispute. The unexpected finding is that among the samples of allied genera compared, Hypermnestra helios appears to be the most closely related to the "Apollos", despite morphological and behavioral dissimilarity. Furthermore, in contrast to the previous higher taxonomy, Archon apollinus which is classified in the tribe Parnassiini was found genetically closer to the tribe Zerynthiini, raising a taxonomic controversy.

Description and molecular phylogeny of a new species of Phoronis (Phoronida) from Japan, with a redescription of topotypes of P. ijimai Oka, 1897.

We describe Phoronis emigi sp. n. as the eighth member of the genus based on specimens collected from a sandy bottom at 33.2 m depth in Tomioka Bay, Amakusa, Japan. The new species is morphologically similar to P. psammophila Cori, 1889, but can be distinguished from the latter by the number of longitudinal muscle bundles in the body wall (56-72 vs. 25-50 in P. psammophila) and the position of the nephridiopores (situated level with the anus vs. lower than the anus in P. psammophila). Using sequences of the nuclear 18S and 28S rRNA genes and the mitochondrial cytochrome c oxidase subunit I (COI) gene, we inferred the relationship of P. emigi to other phoronids by the maximum likelihood method and Bayesian analysis. The analyses showed that P. emigi is closely related to P. hippocrepia Wright, 1856 and P. psammophila Cori, 1889. We describe the morphology of the topotypes and additional material for P. ijimai Oka, 1897. Neither our morphological observations of P. ijimai, nor the phylogenetic analyses based on 18S and COI sequences, contradicts that P. vancouverensis Pixell, 1912 is conspecific with P. ijimai, a synonymy that has long been disputed.