The oldest Gondwanan fossil of Leiinae (Diptera, Mycetophilidae): Phylogenetic and evolutionary implications.

A fossil Mycetophilidae from the Aptian Crato Formation-Cretomanota gondwanica gen. nov., sp. nov.-is described, which is the first mycetophilid from the Crato Formation and corresponds to the oldest known fossil leiine and only the second Gondwanan fossil mycetophilid described so far. Cretomanota gondwanica and both species of Alavamanota Blagoderov and Arillo were added as terminals to the data matrix of a general phylogenetic analysis of the Mycetophilidae, and both fit into the Leiinae. Alavamanota is monophyletic, sister to the clade composed by Cretomanota and the extant genus Manota Williston. The biology of the extant members of this fungivorous family corroborates the reconstruction of the Crato palaeoenvironment as including woodlands with humid habitats and microhabitats. The presence of a Cretaceous member of the tribe Manotini at low latitudes in South America reinforces the hypothesis that the clade with all manotines except Leiella Edwards corresponds to a Lower Cretaceous offshoot from a group in southern Gondwana expanding its distribution to more northern areas into the Gondwana and into Laurasia.

Functional feeding groups of Chironomidae (Diptera: Nematocera) and their spatial variation in an intermittent hill stream (Ventana Stream, Buenos Aires, Argentina).

The feeding habits of Chironomidae in hill streams in the pampean region have not been described. In this study, we analyzed the gut contents of this insect family with an aim at establishing their diet in an intermittent stream in the Ventania-Hill system. We sampled three sites with different habitat characteristics (i. e., altitude, substrate type, and current velocity). Of the total of nine taxa were recorded, the only one present at all sites was Corynoneura sp. 1. The food items found were detritus, diatoms, filamentous chlorophytes, euglenophytes, vascular plants, fungi, and animal remains. Most of the taxa could be classified as gathering collectors, with those having a higher proportion of detritus in the gut contents although the guts of Pentaneura nr. cinerea, Cricotopus sp. 1, and Onconeura analiae included high proportions of animal remains. No differences in the feeding habits were attributable to the location in the stream. This research contributes to our understanding of the trophic habits of Chironomidae in an intermittent hill stream system of South America.

Odorant-binding Protein 10 From Bradysia odoriphaga (Diptera: Sciaridae) Binds Volatile Host Plant Compounds.

Bradysia odoriphaga (Diptera: Sciaridae) is a major insect pest of seven plant families including 30 commercial crops in Asia. The long-term use of chemical pesticides leads to problems such as insect resistance, environmental issues, and food contamination. Against this background, a novel pest control method should be developed. In insects, odorant-binding proteins (OBPs) transport odor molecules, including pheromones and plant volatiles, to olfactory receptors. Here, we expressed and characterized the recombinant B. odoriphaga OBP BodoOBP10, observing that it could bind the sulfur-containing compounds diallyl disulfide and methyl allyl disulfide with Ki values of 8.01 µM and 7.00 µM, respectively. Homology modeling showed that the BodoOBP10 3D structure was similar to that of a typical OBP. Both diallyl disulfide and methyl allyl disulfide bound to the same site on BodoOBP10, mediated by interactions with six hydrophobic residues Met70, Ile75, Thr89, Met90, Leu93, and Leu94, and one aromatic residue, Phe143. Furthermore, silencing BodoOBP10 expression via RNAi significantly reduced the electroantennogram (EAG) response to diallyl disulfide and methyl allyl disulfide. These findings suggest that BodoOBP10 should be involved in the recognition and localization of host plants.

Comparative Hessian Fly Larval Transcriptomics Provides Novel Insight into Host and Nonhost Resistance.

The Hessian fly is a destructive pest of wheat. Employing additional molecular strategies can complement wheat's native insect resistance. However, this requires functional characterization of Hessian-fly-responsive genes, which is challenging because of wheat genome complexity. The diploid Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly and displays phenotypic/molecular responses intermediate between resistant and susceptible host wheat, offering a surrogate genome for gene characterization. Here, we compared the transcriptomes of Biotype L larvae residing on resistant/susceptible wheat, and nonhost Bd plants. Larvae from susceptible wheat and nonhost Bd plants revealed similar molecular responses that were distinct from avirulent larval responses on resistant wheat. Secreted salivary gland proteins were strongly up-regulated in all larvae. Genes from various biological pathways and molecular processes were up-regulated in larvae from both susceptible wheat and nonhost Bd plants. However, Bd larval expression levels were intermediate between larvae from susceptible and resistant wheat. Most genes were down-regulated or unchanged in avirulent larvae, correlating with their inability to establish feeding sites and dying within 4-5 days after egg-hatch. Decreased gene expression in Bd larvae, compared to ones on susceptible wheat, potentially led to developmentally delayed 2nd-instars, followed by eventually succumbing to nonhost resistance defense mechanisms.

Genetic analysis and fine mapping of the gall midge resistance gene Gm5 in rice (Oryza sativa L.).

KEY MESSAGE: The rice gall midge resistance gene, Gm5, confers remarkable antibiosis and is located in the same region on chromosome 12 in three different rice varieties. Fine mapping narrowed this region to a 49-kb segment and identified two candidate genes showing remarkable response to GM infestation. The Asian rice gall midge (GM; Orseolia oryzae; Diptera: Cecidomyiidae) invades rice shoots and forms galls, adversely affecting plant growth and yield production. Thus, the development of resistant varieties through the identification, mapping, and application of GM resistance genes is considered the most efficient strategy for managing this insect. Here, a GM resistance survey of F2 populations derived from intercrosses between resistant rice varieties 'ARC5984,' '570011,' and 'ARC5833' indicated that the resistance gene Gm5 was located on the same chromosomal region in the three varieties. For the initial mapping, three independent F2 mapping populations were developed for the three resistant varieties, and the Gm5 gene was consistently mapped to the same chromosomal region near marker 12M22.6. Fine mapping, which was conducted using the BC1F2 and BC2F2 populations derived from the 9311/ARC5984 cross, narrowed the Gm5 gene region to a 49-kb segment flanked by the markers Z57 and Z64. In the final mapped region, we detected 10 candidate genes, of which six were analyzed for their relative expression. Consequently, two of these genes, Os12g36830 and Os12g36880, showed significantly higher expression in GM-resistant plants than in GM-susceptible plants at 24 and 72 h after GM infestation. Finally, the PCR amplification of markers 12M22.5 and 12M22.6 yielded clear single bands, and these markers were effectively applied for the marker-assisted selection (MAS) of the Gm5 gene. With the developed MAS markers, the fine mapping of this resistance gene will facilitate its map-based cloning and incorporation into insect-resistant rice varieties through breeding.

Carbon dioxide-induced bioluminescence increase in Arachnocampa larvae.

Arachnocampa larvae utilise bioluminescence to lure small arthropod prey into their web-like silk snares. The luciferin-luciferase light-producing reaction occurs in a specialised light organ composed of Malpighian tubule cells in association with a tracheal mass. The accepted model for bioluminescence regulation is that light is actively repressed during the non-glowing period and released when glowing through the night. The model is based upon foregoing observations that carbon dioxide (CO2) - a commonly used insect anaesthetic - produces elevated light output in whole, live larvae as well as isolated light organs. Alternative anaesthetics were reported to have a similar light-releasing effect. We set out to test this model in Arachnocampa flava larvae by exposing them to a range of anaesthetics and gas mixtures. The anaesthetics isoflurane, ethyl acetate and diethyl ether did not produce high bioluminescence responses in the same way as CO2 Ligation and dissection experiments localised the CO2 response to the light organ rather than it being a response to general anaesthesia. Exposure to hypoxia through the introduction of nitrogen gas combined with CO2 exposures highlighted that continuity between the longitudinal tracheal trunks and the light organ tracheal mass is necessary for recovery of the CO2-induced light response. The physiological basis of the CO2-induced bioluminescence increase remains unresolved, but is most likely related to access of oxygen to the photocytes. The results suggest that the repression model for bioluminescence control can be rejected. An alternative is proposed based on neural upregulation modulating bioluminescence intensity.

Genetic diversity of Asian rice gall midge based on mtCOI gene sequences and identification of a novel resistance locus gm12 in rice cultivar MN62M.

The rice gall midge (RGM), Orseolia oryzae (Wood-Mason), is one of the most destructive insect pests of rice, and it causes significant yield losses annually in Asian countries. The development of resistant rice varieties is considered as the most effective and economical approach for maintaining yield stability by controlling RGM. Identification of resistance genes will help in marker-assisted selection (MAS) to pyramid the resistance genes and develop a durable resistance variety against RGM in areas with frequent outbreaks. In this study, a mitochondrial cytochrome oxidase subunit I (mtCOI) was used to analyze the genetic diversity among Thai RGM populations. The phylogenetic tree indicated that the Thai RGM populations were homogeneously distributed throughout the country. The reactions of the resistant rice varieties carrying different resistance genes revealed different RGM biotypes in Thailand. The Thai rice landrace MN62M showed resistance to all RGM populations used in this study. We identified a novel genetic locus for resistance to RGM, designated as gm12, on the short arm of rice chromosome 2. The locus was identified using linkage analysis in 144 F2 plants derived from a cross between susceptible cultivar KDML105 and RGM-resistant cultivar MN62M with single nucleotide polymorphism (SNP) markers and F2:3 phenotype. The locus was mapped between two flanking markers, S2_76222 and S2_419160. In conclusion, we identified a new RGM resistance gene, gm12, on rice chromosome 2 in the Thai rice landrace MN62M. This finding yielded DNA markers that can be used in MAS to develop cultivars with broad-spectrum resistance to RGM. Moreover, the new resistance gene provides essential information for the identification of RGM biotypes in Thailand and Southeast Asia.

Movement Behavior of the Pine Needle Gall Midge (Diptera: Cecidomyiidae).

The movement behavior of the pine needle gall midge (Thecodiplosis japonensis Uchida Et Inouye (Diptera: Cecidomyiidae)), an invasive species in China, was determined by using a tethered flight technique and digital videography in the laboratory. The flight distance, duration, and speed of females were compared at different ages (2-10 h) and ambient temperatures (17, 21, 26, and 30°C). Female flight distance and duration at 26°C were significantly greater than those at 17°C and 21°C. The age of T. japonensis did not significantly affect the three flight characteristics. For females at 2-10 h of age at 26°C and 70% RH, the maximum flight distance was 667.59 m; the longest flight time was 6,222.34 s; and the fastest flight speed was 0.44 m·s-1. For larvae wetted with water, the highest jump was 5.7 cm; the longest jump was 9.6 cm; and the greatest distance moved in 5 min was 27.13 cm, which showed that the active dispersal potential of larvae was very low.

Adhesive latching and legless leaping in small, worm-like insect larvae.

Jumping is often achieved using propulsive legs, yet legless leaping has evolved multiple times. We examined the kinematics, energetics and morphology of long-distance jumps produced by the legless larvae of gall midges (Asphondylia sp.). They store elastic energy by forming their body into a loop and pressurizing part of their body to form a transient 'leg'. They prevent movement during elastic loading by placing two regions covered with microstructures against each other, which likely serve as a newly described adhesive latch. Once the latch releases, the transient 'leg' launches the body into the air. Their average takeoff speeds (mean: 0.85 m s-1; range: 0.39-1.27 m s-1) and horizontal travel distances (up to 36 times body length or 121 mm) rival those of legged insect jumpers and their mass-specific power density (mean: 910 W kg-1; range: 150-2420 W kg-1) indicates the use of elastic energy storage to launch the jump. Based on the forces reported for other microscale adhesive structures, the adhesive latching surfaces are sufficient to oppose the loading forces prior to jumping. Energetic comparisons of insect larval crawling versus jumping indicate that these jumps are orders of magnitude more efficient than would be possible if the animals had crawled an equivalent distance. These discoveries integrate three vibrant areas in engineering and biology - soft robotics, small, high-acceleration systems, and adhesive systems - and point toward a rich, and as-yet untapped area of biological diversity of worm-like, small, legless jumpers.

Development of SNP assays for hessian fly response genes, Hfr-1 and Hfr-2, for marker-assisted selection in wheat breeding.

BACKGROUND: The Hessian fly response genes, Hfr-1 and Hfr-2, have been reported to be significantly induced in a Hessian fly attack. Nothing is known about the allelic variants of these two genes in susceptible (S) and resistant (R) wheat cultivars. RESULTS: Basic local alignment search tool (BLAST) analysis of Hessian fly response genes have identified three alleles of Hessian fly response gene 1 (Hfr-1) on chromosome 4AL and 7DS, and 10 alleles of Hessian fly response gene 2 (Hfr-2) on chromosome 2BS, 2DL, 4BS, 4BL, 5AL and 5BL. Resequencing exons of Hfr-1 and Hfr-2 have identified a single nucleotide polymorphism (SNP) in the lectin domain of each gene that segregates some R sources from S cultivars. Two SNP assays have been developed. The SNP883_Hfr-1 assay characterizes a 'G/A' SNP in Hfr-1, which differentiates 14 Hessian fly R cultivars from S ones. The SNP1294_Hfr-2 assay differentiates 12 R cultivars from S ones. Each of the two SNPs identified in Hfr-1 and Hfr-2 is 'G/A' and resulted in an amino acid change from isoleucine to valine in the lectin domain of the proteins of the alleles in the R cultivars. In addition to the genotype profiles of Hfr-1 and Hfr-2, generated for a set of 249 wheat cultivars which included a set of 39 R cultivars, this study has genotyped the Hessian fly response gene, HfrDrd, and the H32 gene for the wheat germplasm. Resistant cultivars from different origins with one, two, three or four resistance (R) genes in various combinations/permutations have been identified. CONCLUSION: This study has identified allelic differences in two Hessian fly response genes, Hfr-1 and Hfr-2, between S and R cultivars and developed one SNP assay for each of the genes. These two SNP assays for Hfr-1 and Hfr-2, together with the published assays for HfrDrd and the H32 gene, can be used for the selection and incorporation of one or more of these 4 R genes identified in the different R sources in wheat breeding programs.

A Single Swede Midge (Diptera: Cecidomyiidae) Larva Can Render Cauliflower Unmarketable.

Swede midge, Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae), is an invasive pest causing significant damage on Brassica crops in the Northeastern United States and Eastern Canada. Heading brassicas, like cauliflower, appear to be particularly susceptible. Swede midge is difficult to control because larvae feed concealed inside meristematic tissues of the plant. In order to develop damage and marketability thresholds necessary for integrated pest management, it is important to determine how many larvae render plants unmarketable and whether the timing of infestation affects the severity of damage. We manipulated larval density (0, 1, 3, 5, 10, or 20) per plant and the timing of infestation (30, 55, and 80 d after seeding) on cauliflower in the lab and field to answer the following questions: 1) What is the swede midge damage threshold? 2) How many swede midge larvae can render cauliflower crowns unmarketable? and 3) Does the age of cauliflower at infestation influence the severity of damage and marketability? We found that even a single larva can cause mild twisting and scarring in the crown rendering cauliflower unmarketable 52% of the time, with more larvae causing more severe damage and additional losses, regardless of cauliflower age at infestation.

Are midge swarms bound together by an effective velocity-dependent gravity?

Midge swarms are a canonical example of collective animal behaviour where local interactions do not clearly play a major role and yet the animals display group-level cohesion. The midges appear somewhat paradoxically to be tightly bound to the swarm whilst at the same time weakly coupled inside it. The microscopic origins of this behaviour have remained elusive. Models based on Newtonian gravity do, however, agree well with experimental observations of laboratory swarms. They are biologically plausible since gravitational interactions have similitude with long-range acoustic and visual interactions, and they correctly predict that individual attraction to the swarm centre increases linearly with distance from the swarm centre. Here we show that the observed kinematics implies that this attraction also increases with an individual's flight speed. We find clear evidence for such an attractive force in experimental data.

A comparison between the larval eyes of the dimly luminescent Keroplatus nipponicus and the brightly luminescent Arachnocampa luminosa (Diptera; Keroplatidae).

Larvae of the weakly blue-luminescent fungus gnat Keroplatus nipponicus possess on either side of their heads a small black stemmatal eye with a plano-convex lens approximately 25 µm in diameter. In total, 12-14 retinula cells give rise to a centrally fused rhabdom of up to 8 µm in diameter. The rhabdom's constituent microvilli, approximately 70 nm in width, are roughly orthogonally oriented, a requirement for polarization sensitivity. Screening pigment granules are abundant in the retinula cells and measure at least 1 µm in diameter. In comparison with the stemmatal eye of the brightly luminescent Arachnocampa luminosa, that of K. nipponicus is considerably smaller with a poorer developed lens and a rhabdom that is less voluminous, but possesses wider microvilli. Although the larval eye of K. nipponicus can be expected to be functional, as the larvae react to light with a behavioural response, the eyes are probably mainly involved in the detection of ambient light levels and not, as in A. luminosa, also in responding to the luminescence of nearby conspecifics.

Using Next-Generation Sequencing to Detect Differential Expression Genes in Bradysia odoriphaga after Exposure to Insecticides.

Bradysia odoriphaga (Diptera: Sciaridae) is the most important pest of Chinese chive. Insecticides are used widely and frequently to control B. odoriphaga in China. However, the performance of the insecticides chlorpyrifos and clothianidin in controlling the Chinese chive maggot is quite different. Using next generation sequencing technology, different expression unigenes (DEUs) in B. odoriphaga were detected after treatment with chlorpyrifos and clothianidin for 6 and 48 h in comparison with control. The number of DEUs ranged between 703 and 1161 after insecticide treatment. In these DEUs, 370-863 unigenes can be classified into 41-46 categories of gene ontology (GO), and 354-658 DEUs can be mapped into 987-1623 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expressions of DEUs related to insecticide-metabolism-related genes were analyzed. The cytochrome P450-like unigene group was the largest group in DEUs. Most glutathione S-transferase-like unigenes were down-regulated and most sodium channel-like unigenes were up-regulated after insecticide treatment. Finally, 14 insecticide-metabolism-related unigenes were chosen to confirm the relative expression in each treatment by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The results of qRT-PCR and RNA Sequencing (RNA-Seq) are fairly well-established. Our results demonstrate that a next-generation sequencing tool facilitates the identification of insecticide-metabolism-related genes and the illustration of the insecticide mechanisms of chlorpyrifos and clothianidin.

Two new species of Tasiocera Skuse from Baltic amber (Diptera, Limoniidae).

The paper presents very rare fossil record of crane flies. Inclusions in Baltic amber have documented the second evidence of the existence of the genus Tasiocera (Diptera: Limoniidae: Chioneinae) in ancient epochs of Earth's history. Hypotheses were also introduced regarding the environmental preferences of fossil Dasymolopilus in relation to modern representatives of the subgenus. The genus is numerous in species and widespread in the modern fauna, while only one species, Tasiocera (Dasymolophilus) circumcincta, has been known from the fossil record to date. The paper presents an additional description and graphic documentation of this species based on new materials. The discovery of two other species Tasiocera (Dasymolophilus) gorskii sp. nov. and Tasiocera (Dasymolophilus) baltica sp. nov. in Eocene resins documents the occurence of Tasiocera since Eocene. Interestingly, only subgenus Dasymolophilus is represented both in extinct and extant fauna while subgenus Tasiocera is not known from the fossil record. The new discovery is very significant, and the result of the study adds a brick to our understanding of the evolution of this group of insects and their habitat.

First Upper Triassic Diptera (Insecta) from Germany.

First Upper Triassic dipterans (Nematocera) are described based on isolated wings from Fuchsberg near Seinstedt (Sevatian, uppermost Norian of Lower Saxony, Germany): Crivoptychoptera nebrias gen. et sp. nov. (Psychodomorpha, Ptychopteridae), Ptychopteridae incertae sedis, Dohloboyia triassica gen. et sp. nov. (Bibionomorpha, Boholdoyidae) and Chironomidae incertae sedis (Culicomorpha). All taxa under discussion present the oldest occurrences of their families. Geological background and composition of the Upper Triassic insect fauna of Seinstedt are briefly discussed.

Bradysia (Sciara) coprophila larvae up-regulate DNA repair pathways and down-regulate developmental regulators in response to ionizing radiation.

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

Characterization of three glutathione S-transferases potentially associated with adaptation of the wheat blossom midge Sitodiplosis mosellana to host plant defense.

BACKGROUND: Insect glutathione S-transferases (GSTs), a multifunctional protein family, play a crucial role in detoxification of plant defensive compounds. However, they have been rarely investigated in Sitodiplosis mosellana, a destructive pest of wheat worldwide. In this study, we characterized for the first time a delta (SmGSTd1) and two epsilon GST genes (SmGSTe1 and SmGSTe2) and analyzed their expression patterns and functions associated with adaptation to host plant defense in this species. RESULTS: Expression of these SmGST genes greatly increased in S. mosellana larvae feeding on resistant wheat varieties Kenong1006, Shanmai139 and Jinmai47 which contain higher tannin and ferulic acid, the major defensive compounds of wheat against this pest, compared with those feeding on susceptible varieties Xinong822, Xinong88 and Xiaoyan22. Their expression was also tissue-specific, most predominant in larval midgut. Recombinant SmGSTs expressed in Escherichia coli could catalyze the conjugation of 1-chloro-2,4-dinitrobenzene, with activity peak at pH around 7.0 and temperature between 30 and 40 °C. Notably, they could metabolize tannin and ferulic acid, with the strongest metabolic ability by SmGSTe2 against two compounds, followed by SmGSTd1 on tannin, and SmGSTe1 on ferulic acid. CONCLUSION: The results suggest that these SmGSTs are important in metabolizing wheat defensive chemicals during feeding, which may be related to host plant adaptation of S. mosellana. Our study has provided information for future investigation and development of strategies such as host-induced gene silencing of insect-detoxifying genes for managing pest adaptation. © 2023 Society of Chemical Industry.

A new genus and species of gall midge (Diptera: Cecidomyiidae: Cecidomyiidi) with notes on adult cecidomyiid mouthparts and their terminology.

Camillemyia prominens Gagné, gen. n. et sp. n., (Diptera: Cecidomyiidae; Cecidomyiinae) is described for a gall midge from Mexico with an extremely elongate neck and proboscis. The external and internal basic composition of the proboscis of Cecidomyiidae, as exemplified by Cecidomyia resinicola Osten Sacken, is reviewed to help understand the structural changes found in Cam. prominens as compared to those of Contarinia prolixa Gagné & Byers, another species with spectacularly modified mouthparts.

When the past meets the present: the oldest known Bibioninae, and the youngest known Cretobibioninae (Diptera, Bibionidae) from mid-Cretaceous Myanmar amber.

The Diptera family Bibionidae has a rich fossil record from the Tertiary, however little is known about this family in the Mesozoic. Recently, a number of specimens have been found in mid-Cretaceous Myanmar amber, dating from about 99 MYr BP. I report the finding of the oldest known member of the recent subfamily Bibioninae, as well as the youngest known member of the Cretaceous subfamily Cretobibioninae.The Cretobibioninae is represented by Cretobibio burmiticus sp. n., which suggests a wide, possibly cosmopolitan distribution of the recently discovered subfamily Cretobibionidae, the Bibioninae by Protodilophus semispinosus gen. et sp. n. The latter species is probably related to the recent and Tertiary genus Dilophus, but displays some plesiomorphic traits related to the more recent taxa.