This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

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Cox1-based phylogeny of Eastern Palearctic Drunella (Ephemeroptera: Ephemerellidae), description

2of new species, and redescription of D. cryptomeria (Imanishi)

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4

running title: Molecular phylogeny and morphological description of Drunella species

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6

Kei Wakimura1,6, Shin-ichi Ishiwata2, Minami Hamamura3, Yasuhiro Takemon4,7, Kazumi Tanida3,5,

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Koji Inai1, Mikio Kato1,3*891Biology Laboratory, Faculty of Liberal Arts, Sciences and Global Education, Osaka Metropolitan

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University Nakamozu Campus, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan. 2 Department of

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Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimoogino, Atsugi 243-0292, Japan. 3

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Department of Biology, Graduate School of Science, Osaka Metropolitan University Nakamozu

13Campus, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan. 4 Disaster Prevention Research Institute,

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Kyoto University, Gokasho, Uji 611-0011, Japan. 5 Osaka Museum of Natural History, Nagai-Park,

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Higashi-Sumiyoshi-ku, Osaka 546-0034, Japan. 6 Present address: Faculty of Pharmacy, Osaka

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Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi 584-8540, Japan. 7 Present address: Biology

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Laboratory, Faculty of Liberal Arts, Sciences and Global Education, Osaka Metropolitan University

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Nakamozu Campus, 1-1 Gakuencho, Naka-ku, Sakai 599-8531, Japan.

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*Corresponding author: Mikio Kato

Email: mkato@omu.ac.jp

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Abstract

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The diversity of a mayfly genus, Drunella Needham (Ephemeroptera: Ephemerellidae), was24

examined using relationships of the barcode region of the cytochrome c oxidase subunit I (cox1)

25

gene. Two novel species, Drunella campicola Ishiwata sp. nov. and Drunella parvicarnivora

26

Ishiwata sp. nov. were described and Drunella cryptomeria was redescribed. A cox1 dendrogram

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based on the maximum likelihood method consisted of ten clusters of DNA sequences sampled in

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Japan, which were roughly divided into three groups. The first group comprised D. cryptomeria and29

the two newly described species (D. campicola and D. parvicarnivora), the second group included

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D. basalis and D. ishiyamana, and the third group comprised D. trispina, D. triacantha, D. kohnoi,

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and D. sachalinensis. A cryptic lineage of D. trispina was observed and the distance between the two

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D. trispina clades is comparable to that between D. triacantha and each of the two D. trispina clades.

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The inheritance of the mitochondrial genome in these species is yet unclear, and therefore needs to34

be studied further. In the present study, we were able to confirm the distinct lineages based on cox1

35

and aided in the understanding of the taxonomy of the genus Drunella with descriptions of two new36

species.

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Key words: cox1, cryptic lineage, DNA barcoding, DNA taxonomy, Drunella, Ephemeroptera,

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Japan.

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Introduction

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The discrimination of highly diverse aquatic insects has been greatly advanced by DNA-based

44

taxonomy (Ge et al., 2021; Hebert et al., 2003; Morinière et al., 2017; Suh et al., 2019). DNA

45

barcoding using a partial coding region of mitochondrial cytochrome c oxidase subunit 1 (cox1) has

46

been used to characterize mayflies in North America, and the efficacy of species identification has

47

been evident (Ball et al., 2005; Webb et al., 2012). As for aquatic insects in Japan, information on a48

reference collections (mainly mayflies) is available online (http://www.b.s.osakafu-u.ac.jp/~mkato/J-

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amir_home.htm; Wakimura et al., 2016, 2020).

50

The members of the genus Drunella Needham 1905 are known in Asia and North America

51(Allen, 1980; Jacobus & McCafferty, 2008). They tend to be conspicuous and common in clear and52

rapid streams and occur in groups of large numbers of individuals in Japan (Ishiwata and Inada,

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1996). Drunella was studied previously as a species group by McDunnough (1931a, b) and Traver

54(1932), and as a subgenus of Ephemerella Walsh (Needham, 1927). Allen (1980) and Tshernova

55(1972) elevated it to generic rank and Allen (1980) proposed five subgenera. Subsequently, these56

subgenera have been synonymized with the genus Drunella by Jacobus & McCafferty (2008).

57

Additionally, Ishiwata (1987) reviewed the generic characters of Japanese Ephemerellidae based on58

the concepts of Allen & Edmunds (1962) and recorded eight species and a subspecies from Japan59

under the genus Drunella; basalis, cryptomeria, ishiyamana (= yoshinoensis), kohnoi, sachalinensis

60(= bifurcata), trispina, and triacantha. Afterward, seven species (basalis, cryptomeria, ishiyamana,

61

kohnoi, sachalinensis, trispina, and triacantha) were recorded from Japan in the genus Drunella, as

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D. yoshinoensis was a junior synonym of D. ishiyamana, and D. bifurcata was a junior synonym of63

D. sachalinensis (Ishiwata, 2001, 2018b; Ishiwata & Takemon, 2005a).

64

DNA taxonomy of the genus Drunella in Japan and eastern Asia had been investigated by Jo

65& Tojo (2019), and they found two lineages (a major lineage and a cryptic lineage) in D. basalis

66(Imanishi, 1937) and D. ishiyamana Matsumura, 1931, respectively. Independently of their work, itThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
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was preliminarily noted that D. trispina (Uéno, 1928), which was collected in two localities, were68

divided into two distinct lineages (Wakimura et al., 2020). One of which (a lineage of D. trispina) is

69

shared with a minor clade of D. basalis that was analysed by Jo & Tojo (2019).

70

According to Wakimura et al. (2020), two undescribed groups of a spiny crawler mayfly

71

genus, Drunella Needham, had clustered in a DNA-based neighbour-joining phylogenetic tree, both72of which are closely related to Drunella cryptomeria (Imanishi, 1937). Consequently, to resolve the73

complicated DNA taxonomy of the genus Drunella in Japan, we have collected specimens more

74

extensively and conducted molecular phylogenetic analysis of this taxon. In the course of this work,75

we have morphologically described two new species, D. campicola Ishiwata sp. nov. and D.

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parvicarnivora Ishiwata sp. nov. We also re-described D. cryptomeria (Imanishi, 1937).

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Materials and methods

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Collection of specimens for DNA analyses

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Nymphs were collected in a riverbed. Imagoes were caught using an aerial net. Specimens for DNA

81

analyses were stored at 4 °C and maintained alive until processing (for a maximum of 2 d) or

82

preserved in 80% ethanol (for long-term storage), and those for morphological analysis were stored83

in 75% ethanol at ambient temperature. The sampling information for the specimens that were used

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for DNA analysis is summarized in Table 1. Some of the mature nymphs were reared until

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emergence in the laboratory for morphological analyses.

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DNA analyses

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Total genomic DNA was extracted from the whole body (for very small specimens), muscles (wing89

or leg), or larval haemolymph. Dissected tissues were treated with proteinase K (Promega, Madison,90

WI, USA) and extracted using phenol, and then genomic DNA fractions were precipitated with91

ethanol, as previously described (Wakimura et al., 2020). Cox1 was used as the DNA barcode

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and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

92(Hebert et al., 2003), and the target for sequencing was amplified by polymerase chain reaction

93(PCR) using the primer set described by Folmer et al. (1994) and Sensizyme HotStart Taq Premix

94(RBC Bioscience, New Taipei City, Taiwan) as previously described (Wakimura et al., 2016, 2020).

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The amplified products were sequenced using a BigDye Terminator v3.1 and an ABI3730xl DNA

96

Analyzer (Life Technologies, Carlsbad, CA, USA). The sequence data were deposited in the

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International Nucleotide Sequence Database (INSD). The accession numbers of the sequence data98

obtained in the present study as well as those used for the phylogenetic analysis are listed in Tables 1

99

and 2, respectively.

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Sequence comparison and phylogenetic analysis of cox1

102

The sequence data were aligned using the ClustalW algorithm in the BioEdit software package ver.

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7.2.5 (Hall, 1999), and phylogenetic relationships were estimated using PhyML 3.0 (Guindon et al.,1042010) with the Smart Model Selection option (Lefort et al., 2017). The tree was visualized using

105

FigTree ver. 1.4.4 (Rambaut, 2018). A 606 bp homologous region of the cox1 gene (ranging from106

position 53–658 of MK774360) was used for sequence comparison and phylogenetic analysis. We

107sequenced 88 DNA samples that were collected for this study in Japan and included 102 sequences

108

of Drunella (sampled from Japan, Korea, and Russia) that were retrieved from the INSD. The

109

phylogenetic relationships among the total 190 DNA sequences were examined.

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Morphological analyses

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Imagoes, subimagoes, nymphs, and eggs were included in the morphological analysis. All the113

specimens for morphological analyses were preserved and examined in 75% ethanol. Fully

114

developed eggs were obtained from preserved mature female nymphs, female subimagoes, or female

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imagoes. Scanning electron microscopy analysis was performed as described previously (Ishiwata &116

Fujitani 2018). Morphological nomenclature in this work follows that of Edmunds et al. (1976) andThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
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Bauernfeind & Soldán (2012). Egg terminology follows Koss (1968), Koss & Edmunds (1974),

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Ubero-Pascal & Puig (2009), and Ishiwata & Fujitani (2018). The terms for thoracic morphology

119

used in this study follow Kluge (2004). The type series in this study are deposited in the collections

120of the Museum of Nature and Human Activities, Hyogo, Japan (MNHAH). The other specimens

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used in this study are deposited in the authors' collections.

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123

Abbreviations used in the Description section

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For sexes and stages: ♂, male imago; ♀, female imago; ♂s, male subimago; ♀s, female subimago; n,125nymph. For collection depositories: MNHAH, Museum of Nature and Human Activities, Hyogo,

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Japan; KM, the Kyoto University Museum, Kyoto, Japan; no indication, author's collection.

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Taxonomy of the genus Drunella Needham, 1905 in Japan

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Drunella Needham, 1905 (Type species: Ephemerella grandis Eaton, 1884), monotypy.

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Eatonella Needham, 1927 (Type species: Ephemerella doddsii Needham, 1927). Synonymized by

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Jacobus & McCafferty (2008).
Myllonella Allen, 1980 (Type species: Ephemerella coloradensis Dodds). Synonymized by Jacobus& McCafferty (2008).
Tribrochella Allen, 1980 (Type species: Ephemerella trispina Uéno). Synonymized by Jacobus &McCafferty (2008).
Unirhachella Allen, 1980 (Type species: Ephemerella tuberculata Morgan). Synonymized by
Jacobus & McCafferty (2008).

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Diagnosis

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The genus Drunella in Japan can be characterized in the adult male stage by: (1) the terminal

140

segment of the genital forceps is two to three times as long as broad, (2) the long second segment of141

the genital forceps is more or less distinctly bowed, and (3) the penes are without lateral tubercles,

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lobes, or dorsal or ventral spines. The nymphal stage is characterized by: (1) a flat body (especially143

the femora), (2) protrusions in front of the head (except for some), (3) a transverse field of bristles on

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clypeus, (4) tubercles usually present on the ventral margin of the fore femora, (5) a tibial projection

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with a row of small teeth (in part), and (6) the tarsal claws strongly bowed, usually having only a few

146

denticles. The egg is characterized by: (1) an oval form with a polar cap, (2) a smooth chorion,

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without reticulations, covered with fine rectangular maculae (only visible at high magnification

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under scanning electron microscope), and (3) an attachment structure with a single polar cap and a149

multithread-folded with terminal fibre cluster (MTF) (Ubero-Pascal & Puig, 2009). In contrast,

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chorion is covered with reticulations and 1 to 4 tubercles in each reticulation appear in Cincticostella

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Allen and Ephacerella Paclt, while no tubercle in Torleya Lestage and Teleganopsis Ulmer. The

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surface of chorion in Ephemerella Walsh is rough and pitted, with or without reticulations (Ishiwata

153& Fujitani, 2018).

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Note

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Ishiwata (1987) provided the characters of Japanese Drunella referring to the concept proposed by

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Allen & Edmunds (1962) for adults and nymphs. Kluge (2004) had proposed the diagnostic

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characters for the nymphs. In the present study, the diagnoses described for the adult and nymphal

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stages follow the literatures (Allen & Edmunds, 1962; Ishiwata 1987, Kluge 2004). The egg

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characters were described based on Jacobus & McCafferty (2008) and Ishiwata & Fujitani (2018).

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Jacobus & McCafferty (2008) stated that, none of the polyspecific subgenera indicated by Allen

161(1980) were recovered as monophyletic, and they synonymized four nominal subgenera with

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Drunella (sensu stricto). Ogden et al. (2009) considered the genus Drunella to be monophyletic.

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Bauernfeind & Soldan (2012) took a more reserved approach towards the genus level taxonomy of164

Ephemerellidae and emphasized need for more study.

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Results and discussion

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DNA taxonomy of the genus Drunella in Japan

168

Sequence comparisons of cox1 have been successful in distinguishing individual mayfly specimens

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as DNA barcodes (Ball et al. 2005) and multiple species simultaneously (metabarcoding) (Inai et al.

1702020), although the DNA-based phylogenetic trees were occasionally not consistent with the171morphological taxonomy, owing to the highly variable nature of cox1 (Wakimura et al., 2016). A

172

maximum likelihood cox1 phylogeny in the genus Drunella was estimated as shown in Fig. 1. When173

the phylogenetic tree is rooted by Ephemella strigata (Ephemeridae), the genus Drunella is174

monophyletic under the family Ephemerellidae.

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The D. cryptomeria cluster was surrounded by two clusters of yet undescribed groups that176

are designated as Drunella sp. 1 (named Drunella campicola sp. nov. in this study) and sp. 2 (named

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Drunella parvicarnivora sp. nov. in this study) in Fig. 1. The nymph of Drunella sp. 2 had been178

reported to be morphologically identical to D. cryptomeria but lacking a pair of tubercles on the179vertex of head (Hatta & Ishiwata, 1990; Ishiwata, 2000). D. cryptomeria and the two proposed new180

species showed around 88% sequence similarity. Since 99% of the data pairs of the intrageneric-181

interspecific comparison in Ephemeroptera showed less than 94.2% sequence similarity and the182

median sequence similarity for intraspecific comparisons was 98.2% (Inai et al., 2020), D.

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cryptomeria and the two undescribed groups are suggested to belong to different species. The

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morphological description of these two new species and re-description of D. cryptomeria were185

performed (see the Description sections below). The cox1 sequences of D. lepnevae obtained by Jo

186& Tojo (2019) have conformed to a distinct cluster away from D. cryptomeria, as suggested by

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Jacobus & McCafferty (2008).

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Jo & Tojo (2019) have reported that two lineages of D. ishiyamana were found, with the189cluster of D. latipes interposed between the two lineages. Note that D. latipes has been considered to190

be a synonym of D. ishiyamana (Ishiwata, 2018b; Jacobus & McCafferty, 2004). In this study, one

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specimen (sequence ID: 2017-208; a young nymph specimen) was clustered with the clades of D.

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ishiyamana species group (Fig. 1), suggesting the presence of another cryptic lineage of D.

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ishiyamana. Extensive morphological and genetic investigations using matured nymph and adult194

specimens would reveal the classification of this species complex (Jo & Tojo, 2019). Also, two195

distinct lineages of D. basalis were reported (Jo & Tojo, 2019); one (major clade) is neighbouring to196

the D. ishiyamana group, and the other (minor clade) is shared with D. trispina. As for D. trispina,

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two lineages were suggested based on a preliminary analysis of a small number of specimens

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collected from Nara and Okayama Prefectures (Wakimura et al., 2020). In the present analyses, we

199

have examined the specimens that were identified morphologically as D. trispina, and two distinct

200

lineages were confirmed (Fig. 1). One clade of D. trispina (clade I in Fig. 1) comprised specimens

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collected from Kyoto, Nara, and Wakayama Prefectures, and those examined by Jo & Tojo (2019).

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The second clade (clade II in Fig. 1) of D. trispina, which is shared with D. basalis reported by Jo &203

Tojo (2019), comprised specimens from Kanagawa, Kyoto, and Okayama Prefectures. Sequence204

similarity between these two clades (I and II) of D. trispina was approximately 88% and it was much

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less than the median similarity score for the intraspecific comparison in Ephemeroptera cox1 (98.2%,

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as mentioned above). The subpopulations of D. trispina in Kyoto Prefecture, where both clades are

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found, require more extensive examination to elucidate the inheritance of the mitochondrial genome

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in this species group. The examination of additional nuclear genes in a full phylogenetic framework

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would assist in species delimitation for this genus.

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D. sachalinensis and D. kohnoi are closely related as evidenced by the cox1 sequences, but
are morphologically distinguishable (Ishiwata et al., 2018).

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Description of Drunella campicola Ishiwata, sp. nov.

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(Figs. 2.1–2.18)

215[Japanese name: Sato-togemadarakagerou]

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Male imago

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Length (N = 10): Body, 9.2–11.5 mm; forewing, 11.0–12.9 mm; hind wing, 2.7–3.2 mm; caudal

218

filaments, 9.0–13.0 mm.

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Head: Upper part of compound eye dark brown, lower part black.

220

Thorax: Pronotum chocolate brown to blackish brown. Basisternum of prosternum yellowish brown,221with longitudinal carinae; carinae converging anteriorly (maximum width between lateral margins of222

carina more than twice minimum width; Fig. 2.1, upper arrow); furcasternum chocolate brown to223

blackish brown. Mesonotum (Fig. 1.2) chocolate brown to blackish brown; lateroparapsidal sutures

224(LPs) not terminating at medioparapsidal sutures (MPs); scutellum without posterior prolongation

225

and with a pair of membranous posterior lamellae. Mesosternum chocolate brown; basisternum

226

parallel or slightly narrowed posteriorly; furcasternal protuberances parallel (Fig. 2.1, lower arrow).

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Forelegs: Femora dark brown to black; tibiae dark brown to black, about twice as long as femora;

228

tarsi light gray, and ranked in order of 2nd, 3rd, 4th, 5th, and 1st tarsus, based on their lengths.

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Middle and hind legs: Femora dark brown; tibiae dark brown to black; tarsi yellowish brown, more

230

than half length of tibia.

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Forewings (Fig. 2.3): Hyaline; stigmatic area opaque; primary longitudinal veins yellowish brown

232

basally, blackish brown apically; crossvein brown.

233

Hind wings: Hyaline; costal projection rounded (Fig. 2.4, arrow).

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Abdomen: Terga reddish brown to dark brown, with a transverse paler band along each posterior

235

segment. Penes (Fig. 2.5) slightly expanded apico-laterally, with a V-shaped apical median cleft,

236

shaft slightly swollen medially; second segment of genital forceps sharply angled inward and with

237

subapical constriction; terminal segment more than twice as long as broad. Caudal filaments brown238

basally, pale apically, with brown annulation; terminal filament slightly longer than cerci.

239

Female imago

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Length (N = 8): Body, 9.0–11.0 mm; forewing, 11.2–14.5 mm; hind wing, 2.6–4.0 mm; caudal

241

filaments, 9.0–13.9 mm.

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Other features are similar to male imago except for the usual sexual differences and the following

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characters:

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Thorax: Width of prosternal carinae slightly greater than those of male. Mesosternum, basisternum

245

nearly quadrate; furcasternal protuberances wider than those of male. Fore tibiae relatively short,246

only about 1.2 times as long as fore femora.

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Abdomen: Apex of sternum 9 truncate and often with shallowly rounded median emargination.

248

Male subimago

249

Characteristics are similar to male imago except for the duller general colouration and the following

250

characters:

251

Head: Upper part of compound eye reddish brown, lower part black.

252

Thorax: Pronotum chocolate brown to black. Mesonotum chocolate brown without membranous

253

tubercle at junction of mesonotal suture (MNs); pigmented sclerotization on MPs indistinct;

254

scutellum without long posterior prolongation and with a pair of membranous posterior lamellae.

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Mesosternum, basisternum nearly rectangle.

256

Forewings: Brown; longitudinal vein, crossvein and intercalary black.

257

Hind wings: Whitish brown basally, brown apically; vein light brown.

258

Abdomen: Abdominal terga light brown. Caudal filaments subequal to, or slightly shorter than body259

length.

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Female subimago

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As in male subimago except for the usual sexual differences and the following characters:

262

Thorax: Width of prosternal carinae slightly greater than those of male. Width of mesobasisternum

263

subequal to length; furcasternal protuberances wider than those of male.

264

Abdomen: Apex of sternum 9 truncate and often with shallowly rounded median emargination.

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Mature nymph (Fig. 2.6)

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Length (N = 16): Body, 8.5–12.0 mm; caudal filaments, 8.3–10.7 mm.

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Colouration: General colour uniformly brown, with transverse whitish bands on thorax and268

abdomen.

269

Head (Fig. 2.7): Vertex dark brown without tubercles, with lateral genal projections and a broad

270

frontal shelf with short lateral projections; without occipital tubercles (frontal ocellus somewhat

271

protuberant). Clypeus with a transverse field of bristles projecting forwards (Fig. 2.8, arrow).

272

Labrum (Fig. 2.9) with broad, shallow anteromedian emargination. Mandibles (Figs. 2.10–2.11)273

well-developed, with incisive canines; molar surface of left mandible (Fig. 2.10) not parallel to its

274

outer margin (narrower apically).

275

Hypopharynx (Fig. 2.12): Lingua rounded; superlingua rounded, not truncated with a row of hair

276

along anterior margin. Maxillae (Fig. 2.13) not widened apically, with apical canines and apical277

setae, in contrast to genus Cincticostella, where maxillae are not truncated and without a field of

278

long setae (Ishiwata, 2003); maxillary palpi three-segmented, moderately developed, more than 3/4279

as long as galea-lacina.

280

Labium (Fig. 2.14): Glossae wide (about half as long as width of paragolossa), rounded apically,281

lacking apical projection; paraglossae rounded apically; labial palpi three-segmented; submentum282

widened basally.

283

Thorax: prothorax brown (sometimes light gray) without tubercles, mesothorax and metathorax

284

brown without tubercles, with dark spots.

285

Forelegs (Fig. 2.15): Femora yellowish brown, covered with granules, with a transverse ridge

286

basally, two brownish transverse bands (slightly paler in middle), and large and small tubercles on287

ventral (leading) edge (Fig. 2.15); tibiae with brown band medially, with apical projection (Fig. 2.15,

288

arrow) 1/4 length of tarsi; tarsi with two brownish bands basally and apically; tarsal claws sharply

289

bowed inward, with a denticle basally (Fig. 2.16).

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Middle legs: Femora yellowish brown with two brownish transverse bands, basal band darker than291

subapical band; tibiae with brown band medially; tarsi with two brown bands basally and apically;292

tarsal claws sharply bowed inward, with a denticle basally.

293

Hind legs: Colouration of tibiae and tarsi similar to those of middle legs; small spines along outer294

margin, without spines along inner margin; tarsal claws sharply bowed inward, with a denticle

295

basally.

296

Abdomen: Terga 1–8 brown to light brown (terga 6–7 sometimes chocolate brown); terga 9–10

297

chocolate brown; terga 4–9 with a pair of submedian tubercles; these tubercles are small on segments

298

4–5, often barely discernible; terga 3–7 with lamellate, imbricated gills; terga 4–9 with distinct

299

postero-lateral projections. Abdominal sterna brown. Caudal filaments (Fig. 2.6) less than 1/2–2/3 as

300long as body length, with spines at apex of each segment and with heavy intersegmental setae (2–3

301

times as long as segment); terminal filament slightly longer than cerci.

302

Egg (Figs. 2.17–2.18)

303

Size (N = 30): Long axis length with polar cap, 153 µm; short axis length, 96 µm.

304

Oval, with a polar cap; chorion smooth, covered with fine and rectangular maculae (length, ca 2.5

305µm; width, ca 1.0 µm), with MTF and micropyles (Fig. 2.18); micropyle tagenoform type, with306

chorion sperm guide (csg); chorion sperm guide without micropylar rim; micropylar canal subtle,

307

indistinct in some specimens.

308

Diagnosis

309

Male imagoes are distinguishable from other Japanese Drunella species by the shape of the penes,

310

having a broad V-shaped emargination. Female imagoes, subimagoes of both sexes, and eggs of this311

species are indistinguishable from other Japanese Drunella species. The nymphs of this species can

312

be distinguished from all other Japanese Drunella species by lateral genal projections. There are313

certain Nearctic Drunella species that have nymphal genae produced into wide flanges or antero-This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

314

lateral projections (Allen & Edmunds, 1962). In the Japanese Drunella species, D. campicola is the315

only species that has nymphal genae produced into lateral projections, however.

316

Type Material

317

Holotype: female nymph with eggs (in alcohol), labelled, Seiryu Bridge, Kiyotake River, Kiyotake-318

machi, 31 51' 20.8" N 131 23' 34.3" E, 23.III.1988, Miyazaki Prefecture, Kyushu Island, Japan,

319

determined by S. Ishiwata and deposited in MNHAH (MNHAH B2-446671).

320

Paratypes: Two female nymphs with eggs (MNHAH B2-446672, MNHAH B2-446673), same data321

and depository as holotype.

322

Other material examined: [Kyushu Island]: Fukuoka Prefecture: 1n, 21.IV.1988, Tsukushino-shi,

323

Chikugo Riv., Homan Riv., collected by T. Nozaki; 1n, 15.IV.2012, Asakura-shi, Chikugo Riv.,

324

Erizeki, 33°21'47.6"N 130°39'55.4"E, collected by S. Ishiwata; 2n, 16.IV.2012, Yame-shi, Hoshino

325

Riv., Yoriguchi Bridge, 33°11'41.1"N 130°42'50.1"E, collected by S. Ishiwata; 1n, 16.IV.2012,

326

Yame-shi, Yabe Riv., Yabe, 33°11'41.1"N 130°42'50.1"E, collected by S. Ishiwata; 2n, 15.IV.2012,

327

Asakura-shi, Myoken Riv., Sakaai, collected by S. Ishiwata. Saga Prefecture: 2n, 15.IV.2012,

328

Nabeshima-machi, Kakihisa, Tafuse Riv., Sakura Bridge, 33°17'30.9"N 130°16'48.4"E, collected by

329

S. Ishiwata. Miyazaki Prefecture: 1n, 21.IV.1988, Kiyotake-machi, Kiyotake Riv., Seiryu Bridge,

330

31°51'20.8"N 131°23'34.3"E, collected by S. Ishiwata; 6n, 5.II.1988, ibid., Y. Kuroki; 23n,331

23.III.1988, ibid, collected by S. Ishiwata; 3♂, 2♀, 1♂s, 7♀s, 23.III.1988 (reared from nymph.

332

4.IV.–7.V.1988), ibid, collected by S. Ishiwata.

333

Distribution

334

Japan (Kyushu Island: Fukuoka, Miyazaki and Saga Prefectures)

335

Biology336

The nymphs of D. campicola are common in shallow and gently flowing waters of downstream

337

rivers (third-order streams). Although they inhabit diverse types of river bottoms, they are most338

commonly found among gravel bottoms. The life cycle of D. campicola is univoltine-fast, and allThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

339

adults emerge from April to mid-May. The appearance of nymphs and adults from April to June340

suggests that a long egg diapause occurs from summer to winter. The nuptial flight of this species341

has never been observed.

342

Etymology

343

The specific epithet ‘campicola’ derives from nymphal habitat of this species, ‘campus’ and ‘colo’

344

which mean ‘plain’ and ‘habitat’, respectively.

345
346

Description of Drunella parvicarnivora Ishiwata, sp. nov.

347

(Figs. 3.1–3.11)

348[Japanese name: Mukobu-togemadarakagerou]

349

Drunella sp., Hatta & Ishiwata (1990), (nymph, faunal list) [Honshu Island: Aichi Prefecture].

350

Drunella sp., Ishiwata et al. (1991), (nymph, faunal list) [Shikoku Island: Kochi Prefecture,

351

Tokushima Prefecture].

352

Male imago

353

General colour brown with light lateral stripes. Body slender with long caudal filaments.

354

Length (N = 33): Body, 5.0–6.5 mm; fore wing, 6.2–6.5 mm; hind wing, 1.5–1.8 mm; caudal

355

filaments, 10.0–12.0 mm.

356

Head: Colour chocolate brown; upper part of compound eye light brown, lower part black.

357

Thorax: Pronotum chocolate brown to blackish brown. Basisternum of prosternum yellowish brown358

to blackish brown, with longitudinal carinae; carinae slightly converging anteriorly (maximum width359

between lateral margins of carina less than twice minimum width; Fig. 3.1, upper arrow);

360

furcasternum yellowish brown to blackish brown. Mesonotum (similar to Fig. 2.2) chocolate brown361

to blackish brown; LPs not terminating at MPs; scutellum without posterior prolongation and with a362pair of membranous posterior lamellae. Mesosternum chocolate brown; basisternum slightly

363

narrowed posteriorly; furcasternal protuberances parallel (Fig. 3.1, lower arrow).

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364

Forelegs: Brown, sometimes with a pale spot; fore tibiae about twice as long as fore femora; fore365

tarsi ranked in order of 2nd, 3rd, 4th, 5th, and 1st tarsus based on their lengths.

366

Middle and hind legs: Femur with two dark spots basely and sub-apically; tarsi more than half length367

of tibia.

368

Forewings: hyaline; stigmatic area opaque; longitudinal veins brown basally, hyaline apically;

369

intercalary and crossvein pale.

370

Hind wings: hyaline; costal projection pointed (Fig. 3.2, arrow).

371

Abdomen: Abdominal segments 1–3 dark brown, 4–7 translucent, 8–10 dark brown to black. Terga372

dark brown to black, with transverse paler band along each posterior segment. Sterna pale. Apical373

lateral margin of penes not expanded (Fig. 3.3), sides nearly parallel; second segment of genital

374

forceps is slightly distinctly bowed, but not strongly bowed inwards with a deep constriction as in D.

375

basalis (Imanishi, 1937, p. 321, pl. 23, fig. 1); the terminal segment of genital forceps two to three376

times as long as broad. Caudal filaments brown basally, pale apically, with brown annulation;

377

terminal filament slightly longer than cerci.

378

Female imago

379

Length (N = 10): Body, 5.9–6.0 mm; forewing, 5.2–7.0 mm; hind wing, 1.2–2.0 mm; caudal

380

filaments, 5.0–6.2 mm.

381

Other features are similar to male imago except for the usual sexual differences and the following

382

characters:

383

Thorax: Width of prosternal carinae slightly greater than those of male. Mesobasisternum nearly384

quadrate; furcasternal protuberances wider than those of male. Fore tibiae relatively short, only about

385

1.2 times as long as fore femora.

386

Abdomen: Abdominal segments 1–3 dark brown, 4–7 yellowish brown, 8–10 dark brown to black.

387

Terga dark brown to black, with transverse paler band along each posterior segment. Sterna pale.

388

Apex of sternum 9 slightly elongate, not truncate.

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389

Male subimago

390

Characteristics are similar to male imago except for the duller general colouration and the following

391

characters:392

Head: Upper part of compound eye reddish brown, lower part black.

393

Thorax: Pronotum chocolate brown to black. Mesonotum chocolate brown without membranous

394

tubercle at junction of MNs; pigmented sclerotization on MPs indistinct; scutellum without long395

posterior prolongation and with a pair of membranous posterior lamellae. Wings gray; vein dark;

396intercalary and crossvein infuscated.

397

Abdomen: Abdominal segments 4–7 light brown. Caudal filaments subequal to, or slightly shorter

398

than body length.

399

Female subimago

400

Characteristics are similar to male subimago except for the usual sexual differences and the401following characters:

402

Thorax: Width of prosternal carinae slightly greater than those of male. Width of mesobasisternum

403

subequal to length; furcasternal protuberances wider than those of male.

404

Abdomen: Apex of sternum 9 slightly elongate, not truncate.

405

Mature nymph

406

Length (N = 10): Body, 5.2–6.2 mm; caudal filaments, 2.0–3.2 mm.

407

Colouration: General colour yellowish to brown with variable dark brown markings, and with a408transverse blackish band on thorax, legs, and abdomen.

409

Head: Vertex dark brown without tubercles. Genae rounded. Clypeus (similar to Fig. 2.8) with a410

transverse field of bristles projecting forwards. Labrum (similar to Fig. 1.9) with broad, shallow

411

anteromedian emargination. Mandibles (Figs. 3.5–3.6) with narrow incisors on right mandible; molar

412

surface of left mandible not parallel to its outer margin.

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413

Hypopharynx (Fig. 3.7): Lingua rounded; superlingua rounded, with a row of hairs along anterior

414

margin. Maxillae (Fig. 3.8) not widened apically, with apical canines, without an apical tuft of setae,

415

and without a cuticular tooth on medio-anterior edge of galea-lacinia (not as in genus Cincticostella);416

maxillary palpi three-segmented, moderately developed, more than 3/4 as long as galea-lacina.

417

Labium (Fig. 3.9): Glossae wide (approximately 1/2 times as long as width of paragolossa), rounded418

apically, lacking apical projection; paraglossae rounded apically; labial palpi three-segmented;

419

submentum widened basally.

420

Thorax: Prothorax dark brown without tubercles; mesothorax dark brown with variable dark brown

421

markings, with a black band posteriorly (posterior margin of forewing pads); metathorax dark422

brown.

423

Forelegs: Femora yellowish, covered with granules, with two brownish transverse bands (basal

424

bands ridged), and around 15 small tubercles on ventral (leading) edge; tibiae brown medially and425

without apical tibial projection; tarsi light gray basally; tarsal claws (similar to Fig. 2.16) sharply

426

bowed inward, with a denticle basally.

427

Middle legs: Femora yellowish with two brown transverse bands, basal band darker than subapical428

band; tibiae with a brown band medially; tarsi with two brown bands basally and apically; tarsal

429

claws sharply bowed inward, with a denticle basally.

430

Hind legs: Colouration of tibiae and tarsi similar to those of middle legs; small spines along outer431

margin, without spines along inner margin; tarsal claws sharply bowed inward, with a denticle

432

basally.

433

Abdomen: Terga 1–8 yellowish brown (tergum 7 sometimes chocolate brown posteriorly); terga 9–434

10 chocolate brown, terga 1–8 yellowish brown; terga 3–9 with a pair of submedian tubercles; these435

tubercles are small on segments 3–4, often discernible; terga 3–7 with lamellate, imbricated gills;

436

terga 4–9 with distinct postero-lateral projections. Abdominal sterna brown. Caudal filaments lessThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

437

than 1/2–2/3 as long as body length, with brown annulation at apex of each segment, and with short

438

hair-like setae (setae length less than each segment); terminal filament slightly longer than cerci.

439

Egg (Figs. 3.10–3.11)

440

Size (N = 30): Long axis length with polar cap, 154 µm; short axis length, 92 µm.

441

Oval, with a polar cap; chorion smooth, covered with fine and rectangular maculae (length, ca 3.0442µm; width, ca 1.0 µm), with MTF and without rim; micropylar canal subtle, indistinct in some443

specimens.

444

Diagnosis

445

Imagoes, and subimagoes of both sexes and eggs of this species are indistinguishable from that of D.

446

cryptomeria. The nymphs of this species can be distinguished from D.

447

cryptomeria by not having tubercles on the vertex of the head, and by three-segmented maxillary448

palpi. See also the 'Note' below.

449

Type Material

450

Holotype: nymph (in alcohol), labelled, Nikko-shi, Ojika Riv., 36°57'51.2"N 139°41'29.2"E, Tochigi

451

Prefecture, Honshu Island, Japan, 7.VI.2022, determined by S. Ishiwata and deposited in MNHAH

452(MNHAH B2-446674).

453

Paratypes: 4 nymphs (in alcohol; MNHAH B2-446675, 446676, 446677, and 446678), same data454

and depository as holotype.

455

Other material examined: Hokkaido:1n, 28.VI.1993, Atsuta-mura, Atsuta Riv., collected by T. Ito;456

7n, 29.V.1996, Tomakomai-shi, Horonai Riv., collected by H. Miyake; 3n, 21.VII.1987, Tomakomai-

457shi, Yufutsu Riv., Tomakomai-enshurin, collected by R. Kuranishi; 5n, 6.VII.1992, Chitose-shi,

458

Rankoshi Riv., Rankoshi, collected by T. Ito & Y. Nagayasu; 7n, 9.VII.1985, Nakagawa-machi,

459

Teshio Riv., Tomiwa, collected by S. Ishiwata; 1n, 9.VII.1985, Asahi-machi, Nisama Riv.,

460

Daininisama Bridge, 44°05'29.7"N 142°45'47.9"E, collected by S. Ishiwata; 1♀, 12.VII.2012,

461

Sapporo-shi, Toyohira Riv., Misumai, 42°57'38.1"N 141°15'26.6"E, collected by S. Ishiwata.

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462[Honshu Island]: Aomori Prefecture: 4n, 16.V.1987, Shiura-mura, Imaizumi Riv., Imaizumi,

463

collected by S. Sasaki; 1n, 15.V.1987, Imabetsu-machi, Yomouchi Riv., collected by S. Sasaki. Iwate

464

Prefecture: 3n, 21.VI.1993, Daito-cho, Satetsu Riv., Orikaeshi Bridge, collected by S. Ishiwata; 1n,

465

21.VI.1993, Tono-shi, Hayase Riv., Kamigou Bridge, collected by S. Ishiwata; 3n, 21.VI.1993,

466

Miyako-shi, Nagasawa Riv., Shinden Bridge, 39°37'39.8"N 141°54'59.2"E, collected by S. Ishiwata.

467

Ibaraki Prefecture: 1n, 8.VI.1987, Daigo-machi, Kuji Riv., collected by S. Ishiwata. Tochigi

468

Prefecture: 1n, 7.VI.2022, Nikko-shi, Ojika Riv., 36°57'51.2"N 139°41'29.2"E, collected by S.

469

Ishiwata; Saitama Prefecture: 2n, 22.IV.1994, Hannou-shi, Iruma Riv., Ichinose Bridge,

470

35°52'09.8"N 139°15'39.7"E, collected by S. Ishiwata; 5n, 22.IV.1994, Hidaka-shi, Koma Riv.,

471

Kinchaku, collected by S. Ishiwata. Tokyo: 6n, 8.VI.1991, Ome-shi, Kamagafuchi, collected by T.

472

Yamasaki. Kanagawa Prefecture: 2n, 23.VI.1988, Sagamihara-shi, Midori-ku, Hayato Riv.,

473

35°30'27.9"N 139°10'00.3"E, collected by S. Ishiwata; 1n, 15.VI.1989, Yamakita-machi, Sakawa

474

Riv., Shiraishizawa, 35°29'26.2"N 139°03'32.6"E, SI; 2n, 23.VI.1988, Kiyokawa-mura, Nakatsu

475

Riv., Sakaigawa, collected by S. Ishiwata; 1♂s, 27.VI.1997, Isehara-shi, Hinata Riv., Hinatayakushi,

476

35°26'17.5"N 139°15'09.0"E, collected by S. Ishiwata; 5♂, 5♀, sampling by light traps, 30.V.1993,

477

Kiyokawa-mura, Yatarou Riv., Gongen Bridge, 35°28'24.0"N 139°16'13.0"E, collected by S.

478

Ishiwata. Niigata Prefecture: 8n, 2.VI.1986, Asahi-mura, Nagatsu Riv., collected by T. Nozaki.

479

Shizuoka Prefecture: 1n, 2.VI.1989, Shimizu-shi, Okitsu Riv., collected by S. Ishiwata. Shiga

480

Prefecture: 1n, 9.VI.2019, Moriyama-shi, Meta Riv., Moriyama-machi, 35°03'46.7"N

481

135°59'02.7"E, collected by M. Uenishi. Kyoto Prefecture: 1♂s, 2♀, 7.VI.1987, Kyoto-shi,

482

Saihouzi Riv., collected by S. Ishiwata. Osaka Prefecture: 4n, 21.V.2022, Takatsuki-shi, Akutagawa

483

Riv., Hara, 34°55'14.4"N 135°36'10.1"E, collected by T. Fujitani. Wakayama Prefecture: 4n,

484

24.IV.2021, Kudoyama-machi, Nyu Riv., 34°16'43.0"N 135°36'28.0"E, collected by Y. Takemon; 8n,485

21.V.2022, Hashimoto-shi, Kitamata Riv., Hikotani, 34°15'56.5"N 135°38'35.9"E, collected by M.

486

Kato & K. Wakimura; 13n, 29.V.2022, ibid. [Shikoku Island]: Tokushima Prefecture: 10n,This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

487

25.V.1984, Handa-machi, Handa Riv., Banzai, collected by H. Inubuse. [Kyushu Island]: Fukuoka

488

Prefecture: 1n, 13.V.1986, Yoshii-machi, Kose Riv., Enjuji Riv., collected by S. Ishiwata; 1n,489

13.V.1986, Tanushimaru-machi, Kose Riv., collected by T. Nozaki; 1n, 13.V.1986, Fukuoka-shi,

490

Hacho Riv., Hacho Bridge, 33°29'43.4"N 130°17'46.9"E, collected by T. Nozaki. Kumamoto

491

Prefecture: 1n, 12.V.1986, Izumi-son, Kuriki Riv., collected by T. Nozaki.

492

Note

493

Currently, nine species of the genus Drunella have been recognized in Japan. Of these species, the494nymphs of D. parvicarnivora and D. cryptomeria, are distinguishable from other Japanese Drunella

495

nymphs based on the following characteristics: The frontal shelf of the head has no projections; the496

fore femora have no tibial projections; and the caudal filaments have no long, hair-like setae. As for497

adults, the typical characteristics of both species are a costal projection on the hind wing, and long498

caudal filaments (about 1.5–2 times as body length). Most of the other characters are similar to those499

of the other species in the genus Drunella. Of these characteristics mentioned above, having costal

500

process on hind wing in adult stage is not a morphological feature of this genus that has been

501

conventionally pointed out (Kluge, 2004, p.296, as the plesiomorpies of Ephemerella/fg.2: Attenella

502

Edmunds, Drunella, Timpanoga Needham and etc.; Bauernfeind & Soldán, 2012: p.461). Therefore,

503

further research is also needed on Japanese Drunella species.

504

Distribution

505Japan (Hokkaido, Honshu, Shikoku, and Kyushu: except for Okinawa).

506

Biology507

In Kanagawa Prefecture, Drunella parvicarnivora is restricted from piedmont to mountain streams

508(Ishiwata, 2000, 2005, 2018a). The nymphs of D. parvicarnivora are rather common in the shallow

509

and gently flowing waters of second- and third-order streams. Although they inhabit very diverse510

types of river bottoms, they are most commonly found among gravel and sandy bottoms, as is the511

habitat for D. cryptomeria. D. parvicarnivora has never been collected among leaves, debris, or silt.

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512

The life cycle of this species is univoltine-fast, and all adults emerge in June. Since nymphs and513

adults appear only from April to June, a long egg diapause is considered to occur from summer to514

winter.

515

Etymology

516

The specific epithet is derived from the Latin ‘parvus’ and ‘carnivora’ which mean ‘small’ and517‘carnivorous’, respectively, as S. Ishiwata had observed that the nymphs captured the chironomid

518

larva to predate.

519
520

Redescription of Drunella cryptomeria (Imanishi, 1937)

521(Figs. 4.1–4.11)

522[Japanese name: Futakobu-madarakagerou]

523

Ephemerella cryptomeria Imanishi, 1937.

524

Ephemerella (Drunella) cryptomeria; Edmunds (1959).

525

Drunella cryptomeria; Ishiwata, 2001. checklist [Japan (Honshu Island)]; Ishiwata, 2002.

526

nymph, faunal list [Japan (Honshu Island: Kanagawa Prefecture)]; Jacobus &527

McCafferty, 2004. male, nymph [Japan, Korea, Mongolia]; Ishiwata, 2005. male, nymph,528

female subimago [Japan (Honshu Island)]; Ishiwata & Takemon, 2005b. male, nymph,529

key [Japan (Honshu Island)]; Enkhtaivan & Soldán, 2008. faunal list [Mongolia]; Zhou,

530

2013. checklist [China]; Maruyama & Hanada, 2016. male, female, male subimago,

531

female subimago [Japan (Honshu Island: Kyoto Prefecture)]; Wakimura et al., 2016,532

2020. DNA data [Japan (Honshu Island: Kanagawa Prefecture)]; Ishiwata et al., 2018.

533

male, nymph, key [Japan (Honshu Island)]; Ishiwata, 2018a. faunal list [Japan (Honshu

534

Island)].

535
536

Ephemerella (Drunella) bicornis Gose, 1980. Type series: unknown. Type locality: unknown.
Type depository: unknown. Synonymized by Ishiwata (2001).

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537
538

Drunella bicornis; Ishiwata, 1987; Yamasaki, 1986 [Japan (Honshu Island: Tokyo)]; Ishiwata, 2000
[Japan (Honshu Island: Kanagawa Prefecture)].

539

Ephemerella ‘sp. (trispina group); Tanaka, 1966 [Japan (Honshu Island: Tochigi)].

540

Distribution

541

Japan (Honshu, Kyushu: except for Okinawa), China, Korea, and Mongolia. The continental

542

distribution of this species has been reported by Ishiwata (2018b) and Jacobus & McCafferty (2004)

543

but needs to be investigated further.

544
545

Male imago

546

Characteristics identified in the present study are described below in addition to Imanishi (1937).

547

General colour brown with light lateral stripes. Body slender with long caudal filaments.

548

Length (N = 5): Body, 4.5–6.2 mm; forewing, 5.8–6.6 mm; hind wing, 1.0–1.7 mm; caudal

549

filaments, 11.0–12.3 mm.

550

Head: Colour chocolate brown; upper part of compound eye light brown, lower part black.

551

Thorax: Pronotum chocolate brown to blackish brown. Basisternum of prosternum yellowish brown552

to blackish brown, with longitudinal carinae; carinae slightly converging anteriorly (maximum width

553

between lateral margins of carina less than twice minimum width; Fig. 4.1); furcasternum yellowish

554

brown to blackish brown. Mesonotum (similar to Fig. 2.2) chocolate brown to blackish brown; LPs555

not terminating at MPs; scutellum without posterior prolongation and with a pair of membranous

556

posterior lamellae. Mesosternum (similar to Fig. 2.1) chocolate brown; basisternum narrowed

557

anteriorly; furcasternal protuberances parallel.

558

Middle and hind legs: Tarsi is more than half length of tibiae.

559

Hind wings: Hyaline; costal projection pointed (similar to Fig. 3.2).

560

Abdomen: Apical lateral margin of penes not expanded (Fig. 4.2), sides nearly parallel; second

561

segment of genital forceps is slightly distinctly bowed, but not strongly bowed inwards with a deepThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

562

constriction as in D. basalis (Imanishi, 1937, p. 321, pl. 23, fig. 1); terminal segment of genital

563

forceps two to three times as long as broad.

564

Female imago

565

Length (N = 10): Body, 5.2–6.0 mm; forewing, 6.2–6.5 mm; hind wing, 1.4–1.6 mm; caudal

566

filaments, 5.0–6.2 mm.

567

Characteristics are similar to male imago except for the usual sexual differences and the following

568

features:569

Thorax: Width of prosternal carinae slightly greater than those of male. Mesobasisternum nearly570

quadrate; furcasternal protuberances wider than those of male. Fore tibiae relatively short,571

approximately 1.0–1.2 times as long as fore femora. Abdomen: Apex of sternum 9 slightly elongate,

572

not truncate.

573

Male subimago

574

Characteristics are similar to male imago except for the duller general colouration and the following

575

characters:

576

Head: Upper part of compound eye reddish brown, lower part black.

577

Thorax: Pronotum chocolate brown to black. Mesonotum chocolate brown without a membranous

578

tubercle at junction of MNs; pigmented sclerotization on MPs indistinct; scutellum without long579

posterior prolongation and with a pair of membranous posterior lamellae. Forewings gray; hind580

wings more whitish than forewings, costal processes pointed. Vein dark gray.

581

Abdomen: Abdominal terga 1–3 and 9–10 chocolate brown, terga 4–8 yellow brown. Caudal

582

filaments subequal to, or slightly longer than body length.

583

Female subimago

584

Characteristics are similar to male subimago except for the usual sexual differences and the585

following characters:

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and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

586

Thorax: Width of prosternal carinae slightly greater than those of male. Width of mesobasisternum

587

subequal to the length; furcasternal protuberances wider than those of male.

588

Abdomen: Apex of sternum 9 slightly elongate, not truncate.

589

Mature nymph

590

Length (N = 20): Body, 5.0–6.2 mm; caudal filaments, 4.3–6.5 mm.

591

Colouration: General colour light brown to whitish brown, with a transverse blackish band on

592

thorax, legs, and abdomen (Fig. 4.3).

593

Head: Vertex black to chocolate brown, with a pair of tubercles (Figs. 4.4–4.5); genae rounded.

594

Clypeus with a transverse field of bristles projecting forwards (Fig. 4.4, arrow). Labrum (similar to595

Fig. 2.9) with broad, shallow anteromedian emargination. Mandibles (Figs. 4.6–4.7) with narrow596

incisors on right mandible; molar surface of left mandible not parallel to its outer margin.

597

Hypopharynx (similar to Fig. 3.7): Lingua rounded; superlingua with a row of hairs along anterior

598

margin. Maxillae (Fig. 4.8) not widened apically, with apical canines, without an apical tuft of setae,

599

and without a cuticular tooth on medio-anterior edge of galea-lacinia (not as in genus Cincticostella);

600

maxillary palpi two-segmented, not developed, less than a 1/3 as long as galea-lacina.

601

Labium (similar to Fig. 3.9): Glossae wide (about half as long as width of paragolossa), rounded602

apically, lacking apical projection; paraglossae rounded apically; labial palpi three-segmented;

603

submentum widened basally.

604

Thorax: Prothorax dark gray, sometimes whitish to light gray with a pair of blunt tubercles (Fig. 4.5).

605

Mesothorax and metathorax dark brown to black.

606

Forelegs (Fig. 4.9): Femora whitish with a slightly light gray band subapically, a band of transverse

607

granular processes basally, and 10–15 small tubercles on ventral (leading) edge; tibiae light gray608

medially, and without apical tibial projection; tarsi light gray basally; tarsal claws (similar to Fig.

609

2.16) sharply bowed inward, with a denticle basally.

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

610

Middle legs: Femora whitish, with dark gray at basal half; tibiae light gray medially; tarsi light gray611

basally; tarsal claws sharply bowed inward, with a denticle.

612

Hind legs: Colouration of tibiae and tarsi similar to those of middle legs; small spines along outer613

margin, without spines along inner margin; tarsal claws sharply bowed inward, with a denticle

614

basally.

615

Abdomen: Terga 1–3 and 8–10 chocolate brown, terga 4–7 yellowish brown; terga 2–9 with a pair of616

submedian tubercles; these tubercles are small, often barely discernible; terga 3–7 with lamellate,617

imbricated gills; terga 5–9 with distinct postero-lateral projections. Abdominal sterna brown. Caudal

618

filaments whitish brown to white, about 2/3–3/4 as long as body length, with pale annulation at apex619

of each segment, and lacking long, hair-like setae (length of setae less than each segment); terminal

620

filament slightly longer than cerci.

621

Egg (Figs. 4.10–4.11)

622

Size (N = 30): Long axis length with polar cap, 153 μm; short axis length, 96 μm.

623

Oval, with a polar cap; chorion smooth, covered with fine and rectangular maculae (length, ca 2.5624µm; width, ca 1.0 µm), with MTF; micropyles tagenoform type; sperm guide weakly defined,625

without rim; micropylar canal subtle, indistinct in some specimens.

626

Diagnosis

627

D. cryptomeria is indistinguishable from D. parvicarnivora based on the morphology of imaginal628

and subimaginal specimens of both sexes as well as characters of eggs. The nymph of this species is629

discriminated from D. parvicarnivora on the basis of a pair of tubercles on the vertex of head and630

two-segmented maxillary palpi.

631

Type Material

632

Holotype: male. Type locality: Kyoto Prefecture (Kibune), Honshu Island, Japan. Type depository:

633

KM. No type material examined [Holotype not found].

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

634

Other material examined: [Honshu Island]: Aomori Prefecture: 1n, 21.VI.1993, Mogita-mura,

635

Amida Riv., collected by S. Sasaki. Iwate Prefecture: 5n, 21.VI.1993, Rikuzentakada-shi, Yahagi

636

Riv., Koguroyama, collected by S. Ishiwata. Fukushima Prefecture: 3n, 16.V.1988, Koriyama-shi,

637

Abukuma Riv., Ishimushiro Riv., collected by S. Ishiwata; 2n, 15.V.1988, Aizuwakamatsu-shi, Yu

638

Riv., Higashiyama Dam, collected by S. Ishiwata. Tochigi Prefecture: 1n, 6.VI.1987, Nikko-shi, Yu

639

Riv., Uraminotaki, 36°45'14.4"N 39°33'35.3"E, collected by S. Ishiwata. Kanagawa Prefecture:

640

10n, 28.V.2015, Atsugi-shi, Yatarou-zawa, 35°27'33.0"N 139°15'52.7"E, collected by S. Ishiwata;641

1♂, 29.VI.1988, Hadano-shi, Kaname Riv., Genjirou-sawa, 35°26'28.5"N 139°10'03.0"E, collected642

by S. Ishiwata; 5n, 30.V.1988, ibid.; 3♂, 4♀ (reared from nymph, 10.VII.1988), ibid.; 1♂s,

643

14.VI.1996, Hadano-shi, Harutake-sawa, Minoge, 35°25'00.2"N 139°13'49.9"E, collected by S.

644

Ishiwata; 1♂s, 30.V.1988, Yamakita-machi, Sakawa Riv., Shiraishi-zawa, 35°29'26.2"N

645

139°03'32.6"E, collected by S. Ishiwata; 2♂, 2♀ (reared from nymph, 10.VII.1988), ibid.; 1♂s, 1♀s646(reared from nymph, 30.VI.1988), ibid.; 1n, 18.VI.1981, Yamakita-machi, Sakawa Riv., Shiraishi-

647

zawa, 35°29'26.4"N 139°03'32.8"E, collected by S. Ishiwata; 1s♂1s♀ (reared from nymph,648

30.VI.1988), 29.VI.1988, ibid.; 2♀, 29.VI.2022, sampling by light traps, Yamakita-machi, Sakawa-

649

Riv., Tanasawa-bashi, 35°15'52.8"N 135°46'45.1"E, collected by S. Ishiwata. Fukui Prefecture: 5n,

650

23.V.2016, Ono-shi, Kamihanbara, Kuzuryu Riv., Kuzuryu-dam, 35°53'37.8"N 136°47'02.5"E,

651

collected by N. Honda. Yamanashi Prefecture: 1n, 11.VIII.1986, Koshu-shi, Hi Riv., 35°43'30.0"N

652

138°50'34.3"E, collected by T. Nozaki. Shiga Prefecture: 1n, 9.VI.2019, Moriyama-shi, Meta Riv.,

653

35°03'46.7"N 135°59'02.7"E, collected by M. Uenishi. Kyoto Prefecture: 1n, 19.V.1988, Kyoto-shi,

654

Kibune Riv., Azo-dani, collected by Y. Takemon; 3n, 23.V.2021, Kyoto-shi, Kuramakibune-machi,

655

Kibune Riv., Azo-dani, 35°08'00.0"N 135°45'50.0"E, collected by Y. Takemon & M. Kato; 1n,656

21.V.2022, Kyoto-shi, Nomi-cho, Nomi Riv., 35°15'52.8"N 135°46'45.1"E, collected by T. Fujitani.

657[Kyushu Island]: Oita Prefecture: 1n, 22.III.1989, Yufuin-machi, Oita Riv., Yunohira, 33°11'30.4"N

658

131°19'31.6"E, collected by S. Ishiwata.

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

659

Note

660

Although Imanishi (1937) stated that the length of the tarsi is less than half of the tibiae in the hind661

legs of male imagoes (tibia:tarsus ≈ 5:2), the tarsi of the specimens examined in this study were662

longer than half of the tibiae. Moreover, Imanishi (1937) stated that there was a brownish stain at the663base of the forewings, but this character was indistinct in some specimens.

664

Imanishi (1937) described E. cryptomeria based on the male imagoes, and the nymph had

665

remained undescribed. Gose (1980) described E. bicornis based on the nymphs and synonymized E.

666

yoshinoensis under E. cryptomeria. Ishiwata (2001) concluded that E. bicornis and E. cryptomeria

667

were synonymous, based on the investigation of fresh materials obtained by rearing of E. bicornis

668

nymphs collected from the type locality of E. cryptomeria (Kyoto, Kibune River). Ishiwata (2001)

669

also revealed that, E. yoshinoensis, which was synonymized with E. cryptomeria by Gose (1980),

670

should instead be a synonym of D. ishiyamana. Tiunova and Belov (1984) synonymized D. latipes

671

under D. cryptomeria, following the incorrect concept of D. cryptomeria (Gose, 1980). Jacobus and672

McCafferty (2004) placed E. latipes as a synonym of D. ishiyamana in light of the concepts of

673

Ishiwata (2001), based on the material examined. See also, the notes of Ishiwata (2001) and Ishiwata674& Takemon (2005a) under the species names D. cryptomeria and D. ishiyamana, respectively.

675

The synonymy of D. cryptomeria and morphologically similar species, D. lepnevae, D.

676

longipes, and D. fuso (=Drunella fusongensis Su and Gui, 1995), has extensively been discussed.

677

Jacobus & McCafferty (2008) proposed that D. lepnevae and D. cryptomeria are different species

678

and synonymized D. longipes, D. bicornis, and D. fuso under D. lepnevae. We also consider that D.

679

cryptomeria and D. lepnevae are distinct species but D. bicornis to be a synonym of D. cryptomeria

680

as mentioned above. These species, D. cryptomeria (= D. bicornis), D. lepnevae, and D. longipes

681

shared the feature of paired occipital tubercles. The nymphs of D. cryptomeria (=D. bicornis) are

682

distinguishable from the others by its small body length (approximately 5mm in length: 5.0–6.2683mm), and by the two-segmented maxillae palpi. D. fuso was described based on adults collected inThis is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

684

China (Jilin Province) (Su and Gui, 1995). We have not examined any specimen of D. fuso, and the685

original description by Su & Gui (1995) was insufficient as it did not give taxonomic characters to

686

distinguish D. fuso from the other known Asian species. The male imagoes of both D. cryptomeria

687

and D. parvicarnivora are smaller (4.5-6.5 mm in length), than those of above-mentioned species

688(over 9.0 mm in length). Morphological characters in the adults are variable, and the reliable

689

taxonomic characters may appear only in the nymphal stages.

690

Biology691

In Kanagawa Prefecture, D. cryptomeria is restricted to the mountain streams (Ishiwata 2018a). The

692

nymphs of D. cryptomeria are rather common in the shallow and gently flowing waters of first- and693

second-order streams. Although they inhabit diverse types of river bottoms, they are most commonly

694

found among gravel and sandy bottoms, as is the case for D. parvicarnivora, and they have never695

been collected among leaves, debris, or silt. The life cycle of D. cryptomeria is univoltine-fast, and696

all adults emerge from June to July. The appearance of very early instar nymphs in April suggests

697

that a long egg diapause occurs from summer to winter. Mating flights usually take place over water698

at heights ranging from 150–200 cm to as high as the tops of the trees that border the stream. Flights

699

occur from midafternoon to evening on sunny days.

700
701

Acknowledgements

702

We are pleased to acknowledge the help of those who have found the original literature that we

703

needed namely Kluge N. J. (Department of Entomology, Biological Faculty, Saint-Petersburg State

704

University, Saint-Petersburg Russia) and Tiunova T. (Federal Scientific Centre of the East Asia

705

Terrestrial Biodiversity, Far East Branch of the Russian Academy of Sciences, Vladivostok Russia).

706

We thank the collectors listed in the text who kindly gave us the specimens used in this study.

707

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant

708

Number 17K07541 to MK.

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and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

709
710

Disclosure statement711

The authors have no competing interests to disclose.

712
713

References

714

Allen, R. K. (1980). Geographic distribution and reclassification of the subfamily Ephemerellinae

715(Ephemeroptera: Ephemerellidae). In J. F. Flannagan, & K. E. Marshall (Eds.), Advances in

716

Ephemeroptera biology. Proceedings of the Third International Conference on Ephemeroptera717(pp. 71–91). Plenum Press, New York.

718

Allen, R. K., & Edmunds, G. F. (1962). A revision of the genus Ephemerella (Ephemeroptera,

719

Ephemerellidae) V. The subgenus Drunella in North America. Miscellaneous Publications of720

the Entomological Society of America, 3(5), 147–179.

721

Ball, S. L., Hebert, P. D. N., Burian, S. K., & Webb, J. M. (2005). Biological identifications of722

mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Benthological723

Society, 24(3), 508–524. https://doi.org/10.1899/04-142.1

724
725
726

Bauernfeind, E., & Soldán, T. (2012). The mayflies of Europe (Ephemeroptera). Apollo Books,
Ollerup.
Edmunds, G. F. (1959). Subgeneric groups within the mayfly genus Ephemerella (Ephemeroptera:

727

Ephemerellidae). Annals of the Entomological Society of America, 52(5), 543–547.

728

https://doi.org/10.1093/aesa/52.5.543

729
730

Edmunds, G. F., Jensen, S. L., & Berner, L. (1976). The mayflies of North and Central America.
University of Minnesota Press, Minneapolis.

731

Enkhtaivan, S., & Soldán, T. (2008). Mayflies of the Lake Hovsgol region, Mongolia. In F. R. Hauer,

732

J. A. Stanford, & R. L. Newell (Eds.), International advances in the ecology, zoogeography and733

systematics of mayflies and stoneflies (pp. 103–115). University of California Press, Berkeley.

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

734

Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification735

of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

736

Molecular Marine Biology and Biotechnology, 3(5), 294–299.

737

Ge, Y., Xia, C., Wang, J., Zhang, X., Ma, X., & Zhou, Q. (2021). The efficacy of DNA barcoding in738

the classification, genetic differentiation, and biodiversity assessment of benthic

739

macroinvertebrates. Ecology and Evolution, 11, 5669–5681. https://doi.org/10.1002/ece3.7470

740
741
742

Gose, K. (1980). The mayflies of Japanese. (8) Key to families, genera and species. Aquabiology(Kaiyo to seibutsu), 9, 286–288. (in Japanese)
Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W., & Gascuel, O. (2010). New

743

algorithms and methods to estimate maximum-likelihood phylogenies: assessing the744

performance of PhyML 3.0. Systematic Biology, 59(3), 307–321.

745

https://doi.org/10.1093/sysbio/syq010

746
747

Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.

748

Hatta, K., & Ishiwata, S. (1990). Fauna of aquatic insects in the major rivers of the Tokai District,749

central Japan (III). Ephemerellidae (Ephemeroptera) in the major rivers of the Tokai District.

750

The Journal of Nagoya Women’s University, 36, 167–178. (in Japanese)751

http://id.nii.ac.jp/1103/00002120/

752

Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications

753

through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological754

Sciences, 270, 313–321. https://doi.org/10.1098/rspb.2002.2218

755

Imanishi, K. (1937). Mayflies from Japanese torrents VII. Notes on the genus Ephemerella.

756

Annotationes Zoologicae Japonenses, 16(4), 321–329. https://dl.ndl.go.jp/pid/10853520

757
758

Inai, K., Wakimura, K., & Kato, M. (2020). Pairwise sequence comparison data of the DNA barcodesof aquatic insects. Data in Brief, 32, 106284. https://doi.org/10.1016/j.dib.2020.106284

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

759

Ishiwata, S. (1987). Structure and keys of the family Ephemerellidae (1). Structure and keys to760

genera from the family Ephemerellidae. Aquatic Organisms in Kanagawa Prefecture, 9, 27–34.

761(in Japanese)

762
763

Ishiwata, S. (2000). Notes on mayflies in Kanagawa Prefecture. Natural History Report of
Kanagawa, 21, 73–82. (in Japanese)

764

Ishiwata, S. (2001). A checklist of Japanese Ephemeroptera. In Y. J. Bae (Ed.), The 21st century and765

aquatic entomology in East Asia. Proceedings of 1st Symposium of Aquatic Entomology

766

Societies of East Asia (pp. 55–84). The Korean Society of Aquatic Entomology, Seoul.

767

Ishiwata, S. (2002). Mayflies of Kanagawa Prefecture, Japan. Kanagawa-Chûhô Odawara, 138, 1–768

46. (in Japanese)

769

Ishiwata, S. (2003). A revision of the genus Cincticostella (Insecta: Ephemeroptera: Ephemerellidae)

770

from Japan. Species Diversity, 8(4), 311–346. https://cir.nii.ac.jp/crid/1390001204457821696

771
772

Ishiwata, S. (2005). Ephemeroptera. In Insect larvae of Japan (pp. 10–19). Gakken, Tokyo. (inJapanese)773

Ishiwata, S. (2018a). Ephemeroptera. In Kanagawa Entomologists’ Association (Ed.), Insect fauna of774

Kanagawa (pp. 26–44). Kanagawa Entomologists’ Association, Odawara. (in Japanese)

775

Ishiwata, S. (2018b). An annotated catalogue of Japanese Ephemeroptera. Revised edition.

776

Kanagawa Institute of Technology, Division for Environmental Chemistry Research Report, 7,

777

Supplement 1, 1–103.

778

Ishiwata, S., & Fujitani, T. (2018). Eggs of Japanese Ephemeroptera. Kanagawa Institute of779

Technology, Division for Environmental Chemistry Research Report, 7, Supplement 2, 1–63.

780

Ishiwata, S., Furuya, Y., Kuwata, K., & Horiuchi, Y. (1991). Ephemerellidae (Ephemeroptera) in the781

major rivers of Shikoku District. Bulletin of the Biological Society of Kagawa, 18, 19–31. (in782Japanese) http://id.nii.ac.jp/1731/00000947/

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

783
784
785

Ishiwata, S., & Inada, K. (1996). Mayflies common in Japan. Biology of Inland Waters(Rikusuiseibutsugakuho), 11, 36–44. (in Japanese)
Ishiwata, S., & Takemon, Y. (2005a). Checklist of Japanese names of Japanese mayflies with notes786

on nomenclature. Japanese Journal of Limnology, 66(1), 11–35. (in Japanese)787

https://doi.org/10.3739/rikusui.66.11

788

Ishiwata, S., & Takemon, Y. (2005b). Ephemeroptera. In T. Kawai, & K. Tanida (Eds.), Aquatic789

insects of Japan: Manual with keys and illustrations (pp. 31–128). Tokai University Press,

790

Hadano. (in Japanese)

791

Ishiwata, S., Takemon, Y., & Fujitani, T. (2018). Ephemeroptera. In T. Kawai, & K. Tanida (Eds.),

792

Aquatic insects of Japan: Manual with keys and illustrations (2nd ed.) (pp. 47–149, pl. 1–16).

793

Tokai University Press, Hiratsuka. (in Japanese)

794

Jacobus, L. M., & McCafferty, W. P. (2004). Revisionary contributions to the genus Drunella

795(Ephemeroptera: Ephemerellidae). Journal of the New York Entomological Society, 112(2),796

127–147.

797

Jacobus, L. M., & McCafferty, W. P. (2008). Revision of Ephemerellidae genera (Ephemeroptera).

798

Transactions of the American Entomological Society, 134(1), 185–274.

799

https://www.jstor.org/stable/25078993

800

Jo, J., & Tojo, K. (2019). Molecular analyses of the genus Drunella (Ephemeroptera:801

Ephemerellidae) in the East Asian region. Limnology, 20(3), 243–254.

802

https://doi.org/10.1007/s10201-019-00573-3

803
804

Kluge, N. (2004). The phylogenetic system of Ephemeroptera. Kluwer Academic Publishers,
Dordrecht.

805

Koss, R. W. (1968). Morphology and taxonomic use of Ephemeroptera eggs. Annals of the806

Entomological Society of America, 61, 696–721. https://doi.org/10.1093/aesa/61.3.696

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

807

Koss, R. W., & Edmunds, G. F. (1974). Ephemeroptera eggs and their contribution to phylogenetic

808

studies of the order. Zoological Journal of the Linnean Society, 55(4), 267–349.

809

https://doi.org/10.1111/j.1096-3642.1974.tb01648.x

810

Lefort, V., Longueville, J.-E., & Gascuel, O. (2017). SMS: smart model selection in PhyML.

811

Molecular Biology and Evolution, 34, 2422–2424. https://doi.org/10.1093/molbev/msx149

812

Maruyama, H. & Hanada, S. (Eds). (2016). A field guide to Japanese aquatic insects: Adults of813

mayflies, stoneflies and caddisflies. Zenkoku Noson Kyoiku Kyokai Co., Ltd., Tokyo. (in814Japanese)

815

McDunnough, J. (1931a). The bicolor group of the genus Ephemerella with particular reference to816

the nymphal stages (Ephemeroptera). The Canadian Entomologist, 63(2), 30–42, 61–68.

817

McDunnough, J. (1931b). The eastern North American species of the genus Ephemerella and their818

nymphs (Ephemeroptera). The Canadian Entomologist, 63(9), 187–197, 201–216.

819

Morinière, J., Hendrich, L., Balke, M., Beermann, A. J., König, T., Hess, S. K., Müller, R., Leese, F.,

820

Hebert, P. D. N., Hausmann, A., Schubart, C. D., & Haszprunar, G. (2017). A DNA barcode

821

library for Germany’s mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and822

Trichoptera). Molecular Ecology Resources, 17, 1293–1307. https://doi.org/10.1111/1755-

823

0998.12683

824

Needham, J. G. (1927). The Rocky Mountain species of the mayfly genus Ephemerella. Annals of the825

Entomological Society of America, 20, 107–117. https://doi.org/10.1093/aesa/20.1.107

826

Ogden, T. H., Osborne, J. T., Jacobus, L. M., & Whiting, M. F. (2009). Combined molecular and827morphological phylogeny of Ephemerellinae (Ephemerellidae: Ephemeroptera), with remarks

828

about classification. Zootaxa, 1991(1), 28–42. https://doi.org/10.11646/zootaxa.1991.1.2

829
830

Rambaut, A. (2018). Computer program distributed by the author,
https://github.com/rambaut/figtree/releases [accessed 20 January 2022]

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

831

Su, C., & Gui, H. (1995). The first record of the genus Drunella in China with description of a new

832

species (Ephemeroptera: Ephemerellidae). Acta Zootaxonomica Sinica, 20(4), 451–454.

833

Suh, K. I., Hwang, J. M., Bae, Y. J., & Kang, J. H. (2019). Comprehensive DNA barcodes for species

834

identification and discovery of cryptic diversity in mayfly larvae from South Korea:

835

Implications for freshwater ecosystem biomonitoring. Entomological Research, 49, 46–54.

836

https://doi.org/10.1111/1748-5967.12334.

837

Tanaka, H. (1966). Ecological studies on aquatic insects in upper reaches of the Kinu-gawa River,

838

Tochigi Prefecture, Japan. Bulletin of the Freshwater Fisheries Research Laboratory, 15, 123–

839

146.

840
841
842

Traver, J. R. (1932). Mayflies of North Carolina. Journal of the Elisha Mitchell Scientific Society,47(1), 85–161, 47(2), 163–236.
Tiunova, T. M., & Belov, V. V. (1984). On the taxonomy of mayflies (Ephemeroptera,

843

Ephemerellidae) in the south of the Soviet Far East. In I. M. Levanidova, E. A. Makartshenko,844& A. Y. Sementshenko (Eds.), Biology of freshwaters of Far EastUSSR (pp. 74–77). Akademiya

845Nauk SSSR, Dal’nevostochnyi Nauchnyi Tsentr, Vladivostok. (in Russian)

846
847

Tshernova, O. A. (1972). Some new species of mayflies from Asia (Ephemeroptera, Heptageniidae,
Ephemerellidae). Entomologichekoye Obozrenie, 51, 604–614. (in Russian)

848

Ubero-Pascal, N., & Puig, M. A. (2009). New type of egg attachment structure in Ephemeroptera and849

comparative analysis of chorion structure morphology in three species of Ephemerellidae. Acta

850

Zoologica, 90(1), 87–98. https://doi.org/10.1111/j.1463-6395.2008.00367.x

851

Wakimura, K., Takemon, Y., Ishiwata, S., Tanida, K., Abbas, E. M., Inai, K., Taira, A., Tanaka, A.,852& Kato, M. (2020). A reference collection of Japanese aquatic macroinvertebrates. Ecological

853

Genetics and Genomics, 17, 100065. https://doi.org/10.1016/j.egg.2020.100065

854

Wakimura, K., Takemon, Y., Takayanagi, A., Ishiwata, S., Watanabe, K., Tanida, K., Shimizu, N., &855

Kato, M. (2016). Characterization of genes for histone H3, 18S rRNA, and cytochrome oxidase

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

856

subunit I of East Asian mayflies (Ephemeroptera). DNA Barcodes, 4(1), 1–25.

857

https://access.portico.org/stable?au=phx133j8rrk

858

Webb, J. M., Jacobus, L. M., Funk, D. H., Zhou, X., Kondratieff, B., Geraci, C. J., DeWalt, R. E.,

859

Baird, D. J., Richard, B., Phillips, I., & Hebert, P. D. N. (2012). A DNA barcode library for

860

North American Ephemeroptera: progress and prospects. PLos One, 7, e38063.

861

Yamasaki, T. (1986). Ephemeroptera of the Tama River system. In R. Ishikawa (Ed.) Distribution

862

analysis of less migratory insects in Tamagawa area (pp. 79–120). Tokyu Foundation for the863

Better Environment, Tokyo. (in Japanese) https://foundation.tokyu.co.jp/environment/wp-

864

content/uploads/2011/04/b72cfec801650b2f53f631bfddce43f3.pdf

865

Zhou, C. (2013). A species list of Chinese mayflies (Insecta: Ephemeroptera). In K. Tojo, K. Tanida,866& T. Nozaki (Eds.) Proceedings of the 1st Symposium of the Benthological Society of Asia.

867

Biology of Inland Waters, Supplement 2, 167–225.

868

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

869

Legends for Figures

870

Fig. 1. Maximum likelihood cox1 phylogenetic tree of Drunella.

871

The phylogenetic tree is rooted by the outgroup species Ephemera strigata (MN961293). The boot-872

strap reproducibility of the tree topology is indicated at the respective nodes (scores > 50% are

873

shown). The leaves that are labelled with the year and number (part of the specimen identifications)874

were sequenced for this study, and those labelled with the International Nucleotide Sequence Data-875

base (INSD) accession number were retrieved from the INSD. The INSD accession numbers and876

sampling information for the DNA sequences that were sequenced for this study are listed in Table 1.

877

Drunella latipes is considered to be a synonym of D. ishiyamana (as mentioned in the text).

878
879

Fig. 2. Drunella campicola Ishiwata, sp. nov.

880

1–5, male imago: 1, pro- and mesosterna; 2, mesonota; 3, forewing; 4, hind wing; 5, male genitalia

881(dorsal view). 6–16, nymph: 6, mature nymph (dorsal view); 7, head (dorsal view); 8, head (frontal

882view); 9, labrum; 10, left mandible; 11, right mandible; 12, hypopharynx; 13, maxilla; 14, labium;

883

15, foreleg (dorsal view); 16, claw. 17, 18, eggs: 17, general view; 18, chorion. Abbreviations for884

imaginal thorax; BSl, probasisternum; BS2, mesobasisternum; FSl, profurcasternum; FS2,885

mesofurcasternum; LPs, lateroparapsidal suture; MLs, median longitudinal suture; MNs, mesonotal886

suture; MPs, medioparapsidal suture. For eggs; csg, chorion sperm guide; mc, micropylar canal; mo,887

micropylar opening; MTF, multithread-folded with terminal fibre cluster; pc, polar cap.

888
889

This is the accepted manuscript of an article published online by Taylor & Francis in Systematics
and Biodiversity on 29-Aug-2024, available at https://doi.org/10.1080/14772000.2024.2383214

890

Fig. 3. Drunella parvicarnivora Ishiwata, sp. nov.

891

1–3, male imago: 1, pro- and mesosterna; 2, hind wing; 3, male genitalia (dorsal view). 4–9, nymph:

892

4, mature nymph (dorsal view); 5, left mandible; 6, right mandible; 7, hypopharynx; 8, maxilla; 9,893

labium. 10, 11, eggs: 10, general view; 11, chorion. For abbreviations, see the legend of Figure 2.

894
895

Fig. 4. Drunella cryptomeria (Imanishi, 1937).

896

1, 2, male imago: 1, prosterna; 2, male genitalia (dorsal view; Ishiwata & Takemon, 2005b; fig.

89713.6). 3–9, nymph: 3, mature nymph (dorsal view); 4, head (frontal view); 5, head and thorax (lateral

898

view; Ishiwata & Takemon, 2005b; fig. 12.6); 6, left mandible; 7, right mandible; 8, maxilla; 9,899

foreleg (dorsal view). 10, 11, eggs: 10, general view; 11, chorion. For abbreviations, see the legend

900of Figure 2.

Table 1. List of Drunella specimens analysed.
SpeciesDrunella cryptomeria

Sequence ID(Laboratory ID)
2016-027
2016-029
2016-030
2016-031
2016-034
2016-036
2021-232
2021-233
2021-234

MH260770
MH260771
MH260772
MZ820945
MZ820946
MZ820947
MZ820904
MZ820918
MZ820955

Developmental
stage/ sex
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kyoto
Japan: Kyoto
Japan: Kyoto

Sampling dateyear/month/day
2015/5/28
2015/5/28
2015/5/28
2015/5/28
2015/5/28
2015/5/28
2021/5/23
2021/5/23
2021/5/23

INSD accession number

Locality

Drunella campicola sp.nov.

2012-096
2012-179
2012-180
2012-181
2012-182

KF563055
KF563048
KF563049
KF563050
MZ820938

nymph
nymph
nymph
nymph
nymph

missing data
Japan: Fukuoka
Japan: Fukuoka
Japan: Fukuoka
Japan: Fukuoka

missing data
2012/4/15
2012/4/15
2012/4/15
2012/4/15

Drunella parvicarnivora sp. nov.

2011-166
2016-090
2016-092
2016-093
2016-097
2016-107
2016-1082021-063
2021-064
2021-065
2021-066
2021-067

OK501169
MH260779
MH260780
MH260781
MZ820905
MZ820906
MZ820907
OK501170
OK501171
OK501172
OK501173
OK501174

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Japan: Wakayama
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Wakayama
Japan: Wakayama
Japan: Wakayama
Japan: Wakayama
Japan: Wakayama

2011/5/15
2015/6/25
2015/6/25
2015/6/25
2015/6/25
2015/6/25
2015/6/25
2021/4/24
2021/4/24
2021/4/24
2021/4/24
2021/4/24

Drunella basalis

2012-001
2012-002
2012-005
2012-006
2012-007
2012-100
2012-101
2012-160
2012-161
2012-1642013-2412013-274
2017-097
2021-156
2021-157
2021-158

KF563033
KF563034
KF563035
KF563036
KF563037
KF563042
KF563043
KF563044
KF563045
KF563046
MZ820936
MZ820937
MK774308
MZ820916
MZ820923
MZ820903

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
subimago/ female
subimago/ male
nymph
nymph
nymph
nymph

Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Fukuoka
Japan: Fukuoka
Japan: Fukuoka
Japan: Fukuoka
Japan: Fukuoka
Japan: Wakayama
Japan: Wakayama
Japan: Nara
Japan: Kyoto
Japan: Kyoto
Japan: Kyoto

missing data
missing data
missing data
missing data
missing data
2012/4/16
2012/4/16
2012/4/16
2012/4/16
2012/4/16
2013/4/28
2013/4/28
2017/3/18
2021/4/25
2021/4/25
2021/4/25

Drunella ishiyamana

2017-1652017-166
2017-176
2017-1772017-1782017-1792017-1802017-181
2017-1822017-1832017-184
2017-1852017-203
2021-182
2021-184

MK774329
MK774330
MK774333
MK774334
MK774345
MK774346
MK774347
MK774348
MK774349
MK774350
MK774351
MK774352
MK774362
OK501191
OK501193

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Wakayama
Japan: Wakayama

2017/3/18
2017/3/18
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2017/4/22
2021/4/24
2021/4/24

Drunella sachalinensis

2012-054
2012-055
2016-040
2016-043
2021-154
2021-155

KF563040
KF563041
MZ820924
MZ820939
MZ820914
MZ820915

nymph
nymph
nymph
nymph
nymph
nymph

Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Kyoto
Japan: Kyoto

missing data
missing data
2015/5/28
2015/5/28
2021/4/25
2021/4/25

Drunella kohnoi

2017-102
2017-103
2017-104
2017-106
2017-1962017-197
2021-030

MK774396
MK774397
MK774398
MK774401
MK774399
MK774400
MZ820902

nymph
nymph
nymph
nymph
nymph
nymph
nymph

Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Wakayama

2017/3/18
2017/3/18
2017/3/19
2017/3/20
2017/4/22
2017/4/22
2021/4/11

Drunella trispina

2016-037
2016-038
2016-039
2017-1642017-1922017-193
2017-1942017-213
2017-2142021-015
2021-018
2021-095
2021-1642021-167
2021-1682021-177
2021-178

MZ820948
MZ820949
MZ820950
MK774328
MK774359
MK774360
MK774361
MK774382
MK774385
MZ820900
MZ820901
MZ820917
OK501176
OK501178
OK501179
OK501189
OK501190

nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph
nymph

Japan: Kanagawa
Japan: Kanagawa
Japan: Kanagawa
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Nara
Japan: Okayama
Japan: Okayama
Japan: Wakayama
Japan: Wakayama
Japan: Kyoto
Japan: Wakayama
Japan: Kyoto
Japan: Kyoto
Japan: Kyoto
Japan: Kyoto

2015/5/28
2015/5/28
2015/5/28
2017/3/18
2017/4/22
2017/4/22
2017/4/22
2017/6/10
2017/6/10
2021/4/11
2021/4/11
2021/4/25
2021/4/24
2021/4/25
2021/4/25
2021/4/25
2021/4/25

Drunella sp.

2017-208

MK774366

nymphJapan: Nara

2017/4/22

Table 2. List of DNA sequences retrieved from INSD.
Taxon
Sequence ID
INSD accession(Laboratory ID)
number
LC461325
Drunella sp.
n.a.
LC461326
n.a.
LC461327
n.a.
LC461328
D. basalisn.a.
LC461329
n.a.
LC461330
n.a.
LC461331
n.a.
LC461332
n.a.
LC461333
n.a.
LC461334
n.a.
LC461335
n.a.
LC461336
n.a.
LC461337
n.a.
LC461338
n.a.
LC461339
n.a.
LC461340
n.a.
LC461341
n.a.
LC461342
n.a.
LC461343
n.a.
LC461344
n.a.
LC461345
n.a.
LC461346
n.a.
LC461347
n.a.
LC461348
n.a.
LC461349
n.a.
LC461350
n.a.
LC461351
n.a.
LC461352
n.a.
LC461353
n.a.
LC461354
n.a.
LC461355
n.a.
LC461356
n.a.
LC461357
n.a.
LC461358
n.a.
LC461359
n.a.
LC461360
n.a.
LC461361
n.a.
LC461362
n.a.
LC461363
n.a.
LC461364
n.a.
LC461365
n.a.
LC461366
n.a.
LC461367
n.a.
LC461368
n.a.
LC461369
n.a.
LC461370
n.a.
LC461371
n.a.
LC461372
n.a.
LC461373
n.a.
LC461374
n.a.
LC461375
n.a.
LC461376
D. sachalinensis
n.a.
LC461377
n.a.
LC461378
n.a.
LC461379
n.a.
LC461380
n.a.
LC461381
n.a.
LC461382
n.a.
LC461383
n.a.
LC461384
n.a.
LC461385
n.a.
LC461386
n.a.
LC461387
n.a.
LC461388
n.a.
LC461389
n.a.
LC461390
n.a.
LC461391
n.a.
LC461392
n.a.
LC461393
n.a.
LC461394
D. trispina
n.a.
LC461395
n.a.
LC461396
D. kohnoin.a.
LC461397
n.a.
LC461398
D. lepnevae
n.a.
LC461399
n.a.
#1
LC461400n.a.
D. latipes
LC461401
n.a.
LC461402
n.a.
LC461403
n.a.
LC461404
n.a.
LC461405
n.a.
LC461406
D. triacanthan.a.
LC461407
n.a.
LC461408
n.a.
LC461409
n.a.
LC461410
D. ishiyamanan.a.
LC461411
n.a.
LC461412
n.a.
LC461413
n.a.
LC461414
n.a.
LC461415
n.a.
LC461416
n.a.
LC461417
n.a.
LC461418
n.a.
LC461419
n.a.
LC461420
n.a.
LC461421
n.a.
LC461422
n.a.
LC461423
n.a.
LC461424
n.a.
LC461425
n.a.
(outgroup)
MN961293
Ephemera strigata
2013-025
KF563038
Ephemerella atagosana
2012-033
2016-010
MH260767
Ephemerella imanishii #2
Ephemerella notata
2014-023
KP970724
Serratella setigera
JQ655113
2011-605#1#2

Locality

Reference

Russia:Primorskii, ShkotovskiyRayon
Russia:Primorskii, ShkotovskiyRayon
Russia:Primorskii, ShkotovskiyRayon
Japan:Hokkaido, Chitose
Japan:Hokkaido, Isoya
Japan:Yamagata, Yamagata
Japan:Yamanashi, Nirasaki
Japan:Mie, Taiki
Japan:Yamagata, Yamagata
Japan:Iwate, Hanamaki
Japan:Iwate, Hanamaki
Japan:Akita, Yuzawa
Japan:Miyagi, Kami
Japan:Miyagi, Kawasaki
Japan:Miyagi, Shiroisi
Japan:Niigata, Sado
Japan:Fukushima, Aizubange
Japan:Fukushima, Iwaki
Japan:Gunma, Midori
Japan:Tokyo, Hino
Japan:Tokyo, Hino
Japan:Nagano, Shinano
Japan:Nagano, Nagano
Japan:Nagano, Matsumoto
Japan:Nagano, Matsumoto
Japan:Nagano, Matsumoto
Japan:Nagano, Matsumoto
Japan:Toyama, Toyama
Japan:Nagano, Ueda
Japan:Nagano, Ueda
Japan:Nagano, Chikuma
Japan:Nagano, Koumi
Japan:Tochigi, Shioya
Japan:Gifu, Takayama
Japan:Mie, Tsu
Japan:Shiga, Nagahama
Japan:Shiga, Nagahama
Japan:Shiga, Nagahama
Japan:Shiga, Nagahama
Japan:Shiga, Nagahama
Japan:Shiga, Otsu
Japan:Kyoto, Uji
Japan:Nara, Tenkawa
Japan:Osaka, Kaizuka
Japan:Hyogo, Asago
Japan:Hyogo, SayoJapan:Okayama, Maniwa
Japan:Okayama, Maniwa
Japan:Hiroshima, Otake
Japan:Kagawa, Takamatsu
Japan:Miyazaki, Shiiba
Japan:Nagano, Iida
Japan:Okayama, Okayama
Japan:Nagano, Nagano
Japan:Nagano, Azumino
Japan:Niigata, Sado
Japan:Hokkaido, Bibai
Japan:Hokkaido, Horokanai
Japan:Tottori, Tottori
Japan:Nagano, Iida
Japan:Nagano, Matsumoto
Japan:Nagano, Nagano
Japan:Nagano, Nagano
Japan:Tottori, Chizu
Japan:Hokkaido, Horokanai
Japan:Hokkaido, Bibai
Japan:Nagano, Matsumoto
Japan:Hokkaido, Bibai
Japan:Hokkaido, Chitose
Japan:Nagano, Shiojiri
Japan:Nagano, Matsumoto
Japan:Okayama, Maniwa
Japan:Hiroshima, Hiroshima
South Korea:Gangwondo, Injegun
South Korea:Gangwondo, Injegun
South Korea:Gyeongsangnamdo, Sancheonggun
South Korea:Gyeongsangnamdo, Sancheonggun
South Korea:Gyeongsangnamdo, Sancheonggun
South Korea:Gyeongsangnamdo, Sancheonggun
South Korea:Gangwondo, PyeongchangSouth Korea:Gangwondo, PyeongchangSouth Korea:Gangwondo, Jeogeseongun
South Korea:Gangwondo, Jeogeseongun
South Korea:Gyeongsangnamdo, Miryangsi
South Korea:Gyeongsangbukdo, YeongcheonsiJapan:Okayama, Maniwa
Japan:Okayama, Okayama
Japan:Yamaguchi, Iwakuni
Japan:Tottori, Kurayoshi
Japan:Niigata, Iwafune
Japan:Niigata, Iwafune
Japan:Tottori, Kurayoshi
Japan:Okayama, Maniwa
Japan:Nara, Gojo
Japan:Nara, Gojo
Japan:Nagano, Matsumoto
Japan:Tottori, Houki
Japan:Hokkaido, Kamikawa
Japan:Yamanashi, Kai
Japan:Yamanashi, Kai
Japan:Shizuoka, Izu

Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)
Jo and Tojo (2019)Japan:Nara, Yoshino
Japan:Shiga, Takashima
Japan:Kanagawa, Atsugi
Japan:Kyoto, Uji
Japan:Kanagawa, Ashigarakami

Wakimura et al. (2016)
Wakimura et al. (2016)
Wakimura et al. (2020)
Wakimura et al. (2016)
Wakimura et al. (2016)

D. latipes is a synonym of D. ishiyamana as mentioned in the text and no longer valid. This name appears yet in INSD, however.
Ephemerella imanishii was synonymized with Serratella occiprens by Jacobus & McCafferty (2008). This name appears yet in INSD, however.

Outgroup
Drunella lepnevae
Drunella sp. (Russia)
Drunella sp. 1(D. campicola sp. nov.)

100
100
100

76

Kyoto

99

Kanagawa

D. cryptomeria

86

Kanagawa

95

Drunella sp. 2(D. parvicarnivora sp. nov.)

Wakayama

Drunella sp. (Japan)

90

92

D. ishiyamana
60
100

D. latipes #1
59

D. ishiyamana

100

D. basalis

94

D. triacantha

Kyoto
Nara
Wakayama

96

Kanagawa
OkayamaKyoto

96

D. trispina clade I

D. trispina clade II

D. basalis
99

D. kohnoi

D. sachalinensis

86
95

D. kohnoi
D. sachalinensis

0.2