Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing.

BACKGROUND: More than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted based on detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such "molecular operational taxonomic units" (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because well-equipped, well-funded molecular laboratories are not readily available in many biodiverse countries. RESULTS: We here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyperdiverse fly family Phoridae (Diptera). We sequenced 7059 specimens collected in a single Malaise trap in Kibale National Park, Uganda, over the short period of 8 weeks. We discovered > 650 species which exceeds the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3500 barcodes per flowcell. This was achieved by adopting 1D sequencing, resequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; > 99% of specimens) and revealed that 90% of the putative species belong to the neglected, megadiverse genus Megaselia. We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.). CONCLUSIONS: We document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 200,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.

Polyphagy on unpredictable resources does not exclude host specialization: insects feeding on mushrooms.

The degree of ecological specialization plays a crucial role in shaping the structure and functioning of communities. However, comparing specialization within and among groups of organisms is complicated by both methodological issues and conceptual and terminological inconsistencies. Environmental predictability has been considered a key determinant of specialization though empirical evidence is still limited. Fungi and their insect consumers provide a poorly studied but promising system to measure host specialization and test the predictability hypothesis. In this study, we systematically sampled mushrooms in North European boreal forest, and reared total samples of fungivores colonizing the fruitbodies. Due to the unpredictable nature of mushrooms as a resource, low levels of host specialization can be predicted for these insects, which have indeed widely been considered polyphagous. Contrary to expectations, the majority of the studied fungus gnats were found not to exploit their host taxa indiscriminately. Not only were some mushroom taxa never colonized, the infestation rate of acceptable hosts also differed in most of these fungivores. Gnat species themselves formed continua with respect to the estimates of the degree of specialization, derived from parametric individual-based analyses of presence-absence data. In most cases, host use was best explained by models in which the hosts were classified at genus level, with limited support to specialization to particular host species, families, or orders. Indeed, most of the common fungivores appeared to preferentially use various species from one or a few mushroom genera while occasionally feeding on members of other host taxa. This pattern has likely evolved as a compromise between selective forces stemming from host unpredictability and taxon-specific chemical profiles of the mushrooms. Our study highlights the multidimensional nature of ecological specialization: a high number of acceptable hosts does not preclude considerable discrimination among members of the available resource pool. Such situations can only be revealed by individual-based analyses capable of capturing differences in partner-to-partner interaction intensities.

The number of Estonian black fungus gnats (Diptera, Sciaridae) doubled: the first records of 64 species.

Background: Adult sciarid flies are small to medium-sized, delicate insects, typically black in colour and belonging to the superfamily Sciaroidea within the order Diptera. They exhibit a uniform appearance. Distinguishing them from other families of Sciaroidea is primarily achieved through their typical wing venation. Sciaridae are common in both natural and semi-natural ecosystems, where they play a significant role in decomposition during their larval stage. Prior to the current study, only one specific research project had been conducted on Sciaridae in Estonia. The number of sciarid species identified in Estonia was set at 67. New information: This research, conducted in north-eastern Estonia during 2015 and 2016, presents a comprehensive overview of Sciaridae diversity, shedding light on previously understudied aspects of Estonia's biodiversity. A total of 1038 specimens were identified, representing 91 species, amongst which 64 were previously unknown to Estonia. Notably, Corynopterawinnertzi (Mohrig, 1993) emerged as the most abundant species, followed by Corynopterairmgardis (Lengersdorf, 1930), Corynopteracrassistylata (Frey, 1948) and Bradysiatrivittata (Staeger, 1840). The genus Corynoptera Winnertz, 1867 exhibited the highest diversity, consistent with findings from broader European studies. This study underscores the importance of ongoing surveys for better understanding the distribution and composition of Sciaridae species in Estonia, emphasising the need for further research to delve into the intricate ecology and biology of these insects.

New species and new records of Manota Williston (Diptera: Mycetophilidae) in the Neotropical region.

The following 17 new species are described: Manota anfracta (Ecuador), M. appendiculata (Ecuador, French Guyana), M. aristata (Ecuador), M. bisulca (Ecuador), M. depilis (Ecuador), M. inermis (Ecuador), M. iota (Ecuador, French Guyana), M. micula (Ecuador), M. panda (Argentina), M. papillosa (Ecuador), M patula (French Guyana), M. paula (Ecuador), M. pisinna (French Guyana), M. quantilla (Ecuador), M. quantula (Ecuador), M. senta (Nicaragua), M. virgata (Ecuador). New records of the following species are given: Manota acuminata Jaschhof and Hippa (Ecuador), M. acutistylus Jaschhof and Hippa (Ecuador, French Guyana), M. diversiseta Jaschhof and Hippa (Ecuador, French Guyana), M. ibanezi Hippa and Huerta (Ecuador, French Guyana, Peru), M. inornata Jaschhof & Hippa (French Guyana), M. multise tosa Jaschh of and Hippa (Ecuador), M. parva Jaschhof and Hippa (Ecuador), M. rotundistylus Jaschhof and Hippa (Ecuador), and M. squamulata Jaschhof and Hippa (Ecuador).

Morphological and molecular data reveal fifteen new sympatric Zygomyia Winnertz, 1864 (Diptera: Mycetophilidae) species from Ecuador.

Fifteen new Zygomyia Winnertz species are described from material collected from one location in Ecuador during a very short time period: Z. acrotricha sp. nov., Z. comata sp. nov., Z. diversiseta sp. nov., Z. flamma sp. nov., Z. geniculata sp. nov., Z. longistyla sp. nov., Z. napoensis sp. nov., Z. paniculata sp. nov., Z. pectinella sp. nov., Z. spinosa sp. nov., Z. talpaeforma sp. nov., Z. trilobata sp. nov., Z. triseta sp. nov., Z. uniordata sp. nov. and Z. ximoi sp. nov. Zygomyia bicolor Edwards, 1934 is redescribed, including detailed illustration of the male terminalia. The number of Neotropical Zygomyia species is increased to 30. An updated key to the Neotropical Zygomyia is provided. Unexpectedly high level of interspecific genetic variation between Neotropical Zygomyia spp. is reported.

When details matter: Integrative revision of Holarctic Coelophthinia Edwards (Diptera, Mycetophilidae), including mapping of its mitogenome, leads to the description of four new pseudocryptic species.

Background: The small genus Coelophthinia Edwards, 1941 of the subfamily Gnoristinae (Diptera, Mycetophilidae) is so far known to harbour four species from the Palaearctic, Nearctic and Neotropical Regions. Extensive DNA barcoding of fungus gnats of the family Mycetophilidae through the International Barcode of Life project (iBOL) have initiated integrative studies resulting in taxonomic upgrades and a better understanding of many species and their delimitation. The opportunity was also taken to describe the mitogenome of a member of Coelophthinia for the first time. New information: The integrative studies give evidence for splitting the European species C.thoracica Edwards, 1941 into three different species. Four new species are described from the USA, Japan and the Nordic Region in Europe, Coelophthiniacirra Kerr sp. n., Coelophthiniaitoae Kurina sp. n., Coelophthinialata Kjaerandsen sp. n. and Coelophthinialoraasi Kjaerandsen sp. n., raising the number of Holarctic species from two to six. The mitogenome of Coelophthinialoraasi sp. n. is described and analysed.

Pljushtchiaargoi sp. n., a new geometrid moth from the Western Tien Shan Mountains (Lepidoptera, Geometridae, Larentiinae).

Background: This paper focuses on the morphological description and illustration of the wing pattern and genitalia structures of the known species of the genus Pljushtchia. The possibility of co-evolution of Pljushtchia moths and fruit tree forests of Tien Shan is discussed. The maple tree is supposed to have evolved in the Ili River valley in NW China and in Kazakhstan. Malussieversii, the wild apple tree, is distributed in Miocene nemoral forest belt to Europe in the West an to western North America in the East. The last remnants of fruit tree forests are now localised in biodiversity hotspots in China and in Middle Asian mountains. New information: This paper provides a description of a new species of geometrid moth, Pljushtchiaargoi sp. n. As the ancestral Malussieversii has diverged in Malussilvestris in Europe, Malusturkestanica in Kyrgyzstan and Malushissarica in Tajikistan, a co-divergence of geometrid moths and their food plants is possible. We found Chlorissaarkitensis Viidalepp in the Chatkal area, Tien Shan, its sister species Chlorissatalvei Viidalepp in Hissar and Pamirs and Chlorissapretiosaria Staudinger in Transcaucasus. Pljushtchiaprima is associated with a biodiversity hotspot in Tajikistan and Pljushtchiaargoi with another biodiversity hotspot in southern Tien Shan.

Rediscovering the mosquito fauna (Diptera: Culicidae) of Estonia: an annotated checklist with distribution maps and DNA evidence.

Female mosquitoes (Diptera: Culicidae) affect their hosts in numerous negative ways and are crucial to the spread of vector-borne pathogens. It is, therefore, important to have a detailed overview of regional mosquitoes, to be able to detect changes in species diversity and identify possible health threats. The aim of this study was to update the checklist of the mosquito fauna of Estonia for the first time since 1957. For this purpose, 24,344 adult mosquitoes (94% females) were collected in Estonia from 2008 to 2020 using various trapping methods. Specimens were primarily identified by morphological characteristics, but DNA barcoding based on the partial cytochrome c oxidase subunit 1 gene (COI) was also used. Species were included in the checklist based on historical records as well as new collections, while also considering reports from neighboring countries. Species records are supplemented with voucher specimens, distribution maps and DNA evidence. The updated checklist includes 34 species, 27 of which were confirmed with recently collected material. All in all, Aedes communis (de Geer, 1776) proved to be the most common mosquito in Estonia, accounting for 30.1% of the specimens collected. This is noteworthy, as this species has been implicated in the transmission of multiple disease agents present in the area. New evidence revealed the presence of Ae. hexodontus Dyar, 1916, Ae. sticticus (Meigen, 1838), Anopheles messeae Falleroni, 1926 and Culiseta bergrothi (Edwards, 1921) in Estonia.

A contribution towards checklist of fungus gnats (Diptera, Diadocidiidae, Ditomyiidae, Bolitophilidae, Keroplatidae, Mycetophilidae) in Georgia, Transcaucasia.

The fungus gnats of Georgia are studied based on 2682 specimens collected from 57 localities during 2011-2019. Altogether, 245 species are recorded including four species of Bolitophilidae, three species of Diadocidiidae, two species of Ditomyiidae, 34 species of Keroplatidae and 202 species of Mycetophilidae. 230 and 188 species are recorded from Georgia and the whole of Transcaucasia for the first time, respectively. Three new species - Sciophila georgei sp. nov., Leia katae sp. nov. and Anatella metae sp. nov. - are described including detailed illustrations of the male terminalia. Photographs are provided for an additional 38 species to highlight a variability of their general facies. Combined with earlier published data, the number of fungus gnat species in Georgia is set at 246. The estimated diversity of fungus gnats in Georgia is calculated using non-parametric methods and discussed with respect to other Western Palaearctic regions.

Additions to the knowledge of Manota Williston (Diptera: Mycetophilidae) from the Neotropical region, with description of four new species.

The Neotropical species of the genus Manota Williston are studied, based on material of 146 specimens from French Guiana, Ecuador, Nicaragua, Dominica and the Dominican Republic. Four new species are described, viz. M. corniculata sp. n. (French Guiana), M. pseudocavata sp. n. (French Guiana), M. truuverki sp. n. (French Guiana) and M. vladi sp. n. (Dominican Republic). Manota defecta Williston, 1896, the type species of the genus, is listed from Dominica, representing the first record since its description more than a century ago from a Southern Caribbean Island, St. Vincent. New records of 13 additional species are provided: M. acutistylus Jaschhof Hippa, 2005 (Dominica), M. aligera Hippa, Kurina Sääksjärvi, 2017 (French Guiana), M. digitata Hippa, Kurina Sääksjärvi, 2017 (French Guiana), M. iota Hippa Kurina, 2013 (French Guiana), M. micula Hippa Kurina, 2013 (French Guiana), M. nordestina Kurina, Hippa Amorim, 2018 (French Guiana), M. parva Jaschhof Hippa, 2005 (Nicaragua), M. pauloides Hippa, Kurina Sääksjärvi, 2017 (Ecuador), M. perplexa Kurina, Hippa Amorim, 2017 (Nicaragua), M. rotundistylus Jaschhof Hippa, 2005 (Ecuador), M. serrulata Hippa, Kurina Sääksjärvi, 2017 (French Guiana), M. spinosa Jaschhof Hippa, 2005 (French Guiana), M. subaristata Kurina, Hippa Amorim, 2017 (Ecuador). The number of Neotropical Manota species has risen to 96.

Every Single Specimen Counts: A New Docosia Winnertz (Diptera: Mycetophilidae) Species Described from a Singleton.

A new species-Docosia caucasica sp. n.-has been described from material collected from the Lesser Caucasus Mountains in Georgia (Sakartvelo). The new species belongs to a group of Palaearctic species characterized by distinct posterolateral processes of gonocoxites and apically modified setae at the posteroventral margin of the gonocoxites medially. Within the group, D. caucasica sp. n. is most similar to D. landrocki Lastovka and Sevcík, 2006 in having a similar outline of the medial process of posteroventral margin of the gonocoxites and the gonostylus. There is also a marked difference within the partial cytochrome c oxidase subunit 1 gene (COI) sequence of D. caucasica sp. n. and other Docosia spp. available in public databases. As the new species is described from a single male specimen only, the adequacy and code compliance of that are discussed.

Friends in All the Green Spaces: Weather Dependent Changes in Urban Mosquito (Diptera: Culicidae) Abundance and Diversity.

Mosquitoes (Diptera: Culicidae) are universally recognized as troublesome pests and vectors of various pathogens and parasites. Understandably, the species makeup and diversity of individual populations depends on local and broad scale environmental trends, especially on temperature and hydrological variations. Anthropogenic landscapes make for unique habitats, but their effect on insects likely varies across climatic regions. The aim of this study was to investigate the diversity and seasonal patterns of urban mosquitoes in the boreal region. Specimens were collected with an insect net from May to September during three years and determined to species or species group level. Weather information was added to each data point and results analyzed using multivariate regression models. Fieldwork yielded 1890 mosquitoes from four genera. Both abundance and the effective number of species (ENS) significantly decreased during the study period. The number of collected mosquitoes had a negative correlation with wind speed and temperature, latter of which exhibited a negative association with humidity. Species succession followed predictable patterns, but with some variation between years. Still, Culex pipiens/Culex torrentium were the most abundant throughout the study. Importantly, all dominant species were known disease vectors. Our work showed that higher temperatures could result in fewer mosquitoes in boreal towns.

An overview of Estonian woodlice (Isopoda, Oniscidea).

An overview of the Estonian terrestrial isopod fauna is given, based on literature data and material collected from 1984 to 2021. The identified material consisted of 10915 specimens belonging to 14 species and collected from 172 localities throughout Estonia. In combination with previous data from the literature data, there are now reliable records of 16 species of woodlice from Estonia. Two species, viz. Platyarthrushoffmannseggii Brandt, 1833 and Hyloniscusriparius (C. Koch, 1838), are new for the fauna. The latter has probably colonised Estonia recently and range expansions have been reported elsewhere. The data on Philosciamuscorum (Scopoli, 1763) are dubious, and this species is currently excluded from the Estonian list.

An update to the distribution of invasive Ctenolepisma longicaudatum Escherich in northern Europe, with an overview of other records of Estonian synanthropic bristletails (Insecta: Zygentoma).

BACKGROUND: Previously, two species of Zygentoma have been reported as synanthropic in Estonia (Lepisma saccharinum Linnaeus, 1758 and Thermobia domestica (Packard, 1873)). Ctenolepisma longicaudatum Escherich, 1905 is an invasive species that is currently expanding its range in Europe, but had no published records from the northern Baltic Region. NEW INFORMATION: Ctenolepisma longicaudatum was first found in Estonia in 2018. It has currently several established populations in public buildings in Tartu and Tallinn, but has not been found in private households, nor in other places in Estonia. A brief overview of its invasion history in northern Europe is given.

Twenty years of Dipterology through the pages of Zootaxa.

We present a summary and analysis of the Diptera-related information published in Zootaxa from 2001 to 2020, with a focus on taxonomic papers. Altogether, 2,527 papers on Diptera were published, including 2,032 taxonomic papers and 1,931 papers containing new nomenclatural acts, equivalent to 22% of all publications with new nomenclatural acts for Diptera. The new nomenclatural acts include 7,431 new species, 277 new genera, 2,003 new synonymies, and 1,617 new combinations. A breakdown by family of new taxa and new replacement names proposed in the journal during the last two decades is provided, together with a comparison of Zootaxa's output to that of all other taxonomic publications on Diptera. Our results show that the journal has contributed to 20% of all biodiversity discovery in this megadiverse insect order over the last 20 years, and to about 31% in the last decade.

Three new species and new records of Clastobasis Skuse (Diptera: Mycetophilidae) from Japan and the Kuril Islands.

Three new Clastobasis Skuse species are described: C. ainuorum sp. n. (Kuril Islands, Iturup), C. kanabo sp. n. (Japan, Honshu) and C. menguali sp. n. (Japan, Honshu). The new species are figured, photographed and their systematics discussed. New records are provided for C. loici Chandler and C. subalternans Zaitzev, both from Japan (Honshu). The number of Palaearctic Clastobasis species is set up at eight including six of them restricted only to the Eastern Palaearctic. Clastobasis loici, previously known only from Europe, is described to have a Trans-Palaearctic distribution. A key to the Palearctic species of Clastobasis is provided and the distribution shortly discussed.

New species and new records of Sciophila Meigen (Diptera: Mycetophilidae) from the Afrotropical region.

A comprehensive material of Afrotropical Sciophila including 262 male specimens of 15 species collected from 7 countries are studied. Two new species-S. geiri sp. n. and S. tchabalensis sp. n.-are described from Madagascar and Cameroon, respectively. New records of the following 13 species are presented: S. digitilenta Søli, 1997 (Uganda), S. fenestralis Søli, 1997 (South Africa), S. kakumensis Søli, 1997 (Cameroon, Uganda), S. kjaerandseni Søli, 1997 (Uganda), S. koundensis Søli, 1997 (Uganda), S. leptosoma Søli, 1997 (Democratic Republic of Congo), S. longistyla Søli, 1997 (South Africa), S. mazumbaiensis Søli, 1997 (Uganda), S. ocreata Philippi, 1865 (France: La Réunion), S. papula Søli, 1997 (Democratic Republic of Congo), S. pinniger Søli, 1997 (South Africa, Cameroon, Uganda, Kenya), S. quadra Søli, 1997 (Democratic Republic of Congo), S. stellata Søli, 1997 (Uganda). The majority of the new records represent the first ones since initial description of the species. S. ocreata is considered to be introduced to the Island of La Réunion. The number of Afrotropical Sciophila species is set at 23.

Additions to the Estonian fauna of mycophagous Cecidomyiidae (Diptera), with a description of Unicornella estonensis gen. et sp. nov.

Mycophagous gall midges from two Malaise trapping campaigns in Estonia were subjected to a morpho-taxonomic study, with the aim of identifying previously unrecorded species. Fifty-four species are shown for the first time to occur in Estonia, specifically 1 Lestremiinae, 21 Micromyinae, 5 Winnertziinae, and 27 Porricondylinae. This raises the number of mycophagous cecidomyiids known from Estonia to 152, which is about one fourth of the total estimated for the national fauna. One of the Porricondylinae turned out to be new to science and is described as Unicornella estonensis Jaschhof Sikora. Unicornella, a new genus of the tribe Porricondylini, is characterized by that males have a single gonocoxal process, as opposed to two or no processes found in other Porricondylini.

Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region.

The Ochlerotatus (Oc.) communis complex consist of three Northern American species as well as a common Holarctic mosquito (Diptera: Culicidae) Oc. communis (De Geer, 1776). These sister species exhibit important ecological differences and are capable of transmitting various pathogens, but cannot always be differentiated by morphological traits. To investigate the Oc. communis complex in Europe, we compared three molecular markers (COI, ND5 and ITS2) from 54 Estonian mosquitoes as well as two COI marker sequences from Sweden. These sequences were subjected to phylogenetic analysis and screened for Wolbachia Hertig and Wolbach symbionts. Within and between groups, distances were calculated for each marker to better understand the relationships among individuals. Results demonstrate that a group of samples, extracted from adult female mosquitoes matching the morphology of Oc. communis, show a marked difference from the main species when comparing the mitochondrial markers COI and ND5. However, there is no variance between the same specimens when considering the nuclear ITS2. We conclude that Oc. communis encompasses two distinct mitochondrial DNA lineages in the Nordic-Baltic region. Further research is needed to investigate the origin and extent of these genetic differences.

The first Nearctic record of the genus Neocheiridium (Pseudoscorpiones: Cheiridiidae), with description of Neocheiridium gullahorum sp. n.

BACKGROUND: The genus Neocheiridium Beier, 1932 currently contains seven Neotropical (including Caribbean) and two Afrotropical species, with no Nearctic records. NEW INFORMATION: An undescribed species of Neocheiridium from South Carolina was discovered in the Ohio State University Acarology Collection and is described as Neocheiridium gullahorum, n. sp. (based on specimens of both sexes). A key to known world species of Neocheiridium is proposed.