The Imaginal Characters of Neoephemera Projecta Showing Its Plesiomorphic Position and a New Genus Status in the Family (Ephemeroptera: Neoephemeridae)

Article The Imaginal Characters of Neoephemera projecta Showing Its Plesiomorphic Position and a New Genus Status in the Family (Ephemeroptera: Neoephemeridae)

Zhenxing Ma and Changfa Zhou *

College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; [email protected] * Correspondence: [email protected]; Tel.: +86-139-5174-7595

Simple Summary: The phylogenetically problematic Neoephemeridae is a small family of the order Ephemeroptera, including four genera reported previously. They are genera Neoephemera (in Nearctic region), Ochernova (Central Asia), Leucorhoenanthus (West Palearctic) and Potamanthellus (East Palearctic and Oriental regions), which were geographically isolated until the Neoephemera projecta Zhou and Zheng, a species reported in 2001 from Southwestern China, connected them together. In this work, the imaginal stage and biology of Neoephemera projecta are first observed and described. Furthermore, a series of autapomorphies and plesiomorphies of it are recognized and discussed. Morphologically and biologically, this species is significantly different from other genera and deserves a new plesiomorphic position in the Family Neoephemeridae. Therefore, a new genus Pulchephemera gen. n. is established to reflect its primitive position and intermediate characters of two clades of the family. In addition, some shared characters of this new genus and the family Ephemeridae provide a new perspective or possibility on the phylogeny of Neoephemeridae within Citation: Ma, Z.; Zhou, C. The the order Ephemeroptera. Imaginal Characters of Neoephemera projecta Showing Its Plesiomorphic Abstract: The newly collected imaginal materials of the species Neoephemera projecta Zhou and Zheng, Position and a New Genus Status in the Family (Ephemeroptera: 2001 from Southwestern China, which is linking the other genera of the family Neoephemeridae, Neoephemeridae). Insects 2021, 12, are described in detail. Nymphs are also photographed for the first time. The morphology of this 723. https://doi.org/ species shows some characters of the other genera in Neoephemeridae and several autapomorphies. 10.3390/insects12080723 However, most characters can be seen as plesiomorphies of the family. Specifically, the dorsal-oriented fimbriate gills, projected frons and slim labial palpi in nymphs plus large reddishly pigmented Academic Editor: Laurence wings, many crossveins, 4-segmented forceps with a relatively long basal segment, fused penes and A. Mound unforked anal vein show that this species is closer to the taxon Fossoriae rather than to the previously considered Potamanthidae. To reflect its primitive position, a new genus, Pulchephemera Zhou gen. Received: 29 June 2021 n., is established for this species, Pulchephemera projecta comb. n. Its eggs and observed biology are Accepted: 8 August 2021 also described. Published: 12 August 2021

Keywords: Pulchephemera gen. n.; new genus; new combination; phylogeny; mayfly; China Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction The mayfly family Neoephemeridae (Insecta: Ephemeroptera) includes 13 extant species [1–4]. Most species have been described from both imaginal and nymphal materials [1,3,5,6]. An exception is the species Neoephemera projecta Zhou and Zheng, 2001, Copyright: © 2021 by the authors. Neoephemera Licensee MDPI, Basel, Switzerland. the single species of the genus McDunnough, 1925 found in China and the This article is an open access article Eastern Palearctic region, which was originally reported based on nymphs only [2,7]. distributed under the terms and Bae and McCafferty [1] reviewed the family Neoephemeridae and differentiated three conditions of the Creative Commons genera within it: Neoephemera, Ochernova Bae and McCafferty, 1998 and Potamanthellus Attribution (CC BY) license (https:// Lestage, 1931 [8]. In this revision, genus Leucorhoenanthus Lestage, 1931 (including one Euro- creativecommons.org/licenses/by/ pean species L. maximus (Joly, 1871)) was regarded as a synonym of Neoephemera (including 4.0/). the four Nearctic species at that time) and a new genus Ochernova was established for the

Insects 2021, 12, 723. https://doi.org/10.3390/insects12080723 https://www.mdpi.com/journal/insects Insects 2021, 12, 723 2 of 16

Central Asian species Neoephemera tshernovae Kazlauskas, 1963 [9]. However, the genus classification of Neoephemeridae is still controversial. In the system of Kluge [5], L. maximus was still retained as a monospecific taxon Leucorhoenanthus/g1 (= genus Leucorhoenanthus) and this system shows the morphological and zoogeographic distribution gap between Nearctic and Palearctic neoephemerid species. It is believed that the imagoes of N. projecta will provide new evidence for the theory of generic classification in Neoephemeridae. Based on the phylogeny of extant Neoephemeridae in Bae and McCafferty [1], the four genera (sensu Kluge [5]) of Neoephemeridae can be divided into two distinct groups: (1) genus Potamanthellus with vestigial forceps and pigmented wings; (2) Neoephemera-like group (including genera Neoephemera, Ochernova and Leucorhoenanthus), with relatively normal forceps and transparent wings. As the single extant species linking the two groups geographically, the phylogenetic position of N. projecta within Neoephemeridae is very important for the generic phylogeny of this family. Generally, the morphology of the family Neoephemeridae shows an unstable phylogenetic position and enigmatic origin [5,10,11]. Previously, its adults were regarded as possessing potamanthid-like wing venation and genitalia but its nymphs with caenid-like body and gill pattern [12,13]. As the largest species of this family (with large body size and many plesiomorphies), the detailed description of N. projecta will help us test the existing hypothesis or propose a new one for the systematic position of Neoephemeridae. In order to clarify the position of N. projecta, we tried several times to collect the adults of it. Fortunately, in April 2021, tens of males, females and mature nymphs were found and caught by the authors, allowing us to improve our understanding of this species, genus and family. After careful examination, a new genus is established here to include this species and to reflect its plesiomorphic position combining characters of Potamanthellus and Neoephemera.

2. Materials and Methods The nymphs of N. projecta were collected by hand net, and the adult (including live subimagoes and female imagoes) were collected in and near the creek in the afternoon, all male and most female imagoes were reared from subimagoes indoors. All specimens were stored in ethanol (more than 80%). The materials were examined under a stereomicro- scope and photographed with the digital camera system connected with the microscope. Some small structure of nymphs (e.g., setae on mouthpart, gill, terga and femora), subimagoes (tarsal segments) and eggs (dissected from a female imago) were photographed by an SEM (Scanning Electron Microscope). Diagnostic characters and terminology were adopted from Bae and McCafferty [1] and Kluge [5,14]. Abbreviation for wing veins: “C”: Costa; “Sc”: Subcosta; “MA”: Medius anterior; “MP”: Medius posterior; “Rs”: Radial sector; “Cu”: Cubitus; “CuA”: Cubitus anterior; “CuP”: Cubitus posterior; “A”: Anal vein. In addition, considering the unique nymphal characteristics (the mouthparts and the caudal ﬁlament) and lack of adult information [4], the Vietnamese species Potamanthellus unicutibius Nguyen and Bae, 2004 is not mentioned and discussed in this article (including the genetic diagnosis and phylogeny analysis). It seems that the species is a new genus and more information is needed to resolve the confusion. Sample sites of all other materials mentioned in this article: N. purpurea (Traver, 1931) [15]: two nymphs, South Carolina, no other data; N. youngi Berner, 1953 [16]: two male imagoes three female imagoes, FLORIDA, Okaloosa Co., Blackwater River, 9–11 May 1998, WL & J Peters; 10 nymphs, FLORIDA: Walton Co., Gum Cr. on Hwy. 331, 6 mi. NW. of DeFuniak Springs, 24-IV-1967, W. L. & J. Peters; P. chinensis Hsu, 1936 [17]: 20 male imagoes, 20 female imagoes, 30 nymphs, Dishui Lake, Yanqing County, Beijing, 17–23-vi-2018, ZHANG Wei & MA Zhen-Xing. All materials mentioned in the research are deposited in the Mayﬂy Collection, College of Life Sciences, Nanjing Normal University (NNU), China. Insects 2021, 12, 723 3 of 16

3. Results Pulchephemera Zhou, gen. n. Type species and species included: Pulchephemera projecta (Zhou and Zheng, 2001). Mature nymph: body longer than 16.0–20.0 mm, generally flat and stout (Figure 1); head with genal and frons projections (Figure 2A), anterior margin of frons concave; lead- ing margin of labrum slightly concave (Figure 2B); maxillary and labial palpi 3-segmented, relatively slender (Figure 2F,G); anterolateral angles of pronotum and mesonotum extended into clear projections; pronotum extended laterally to same level of mesothoracic projections (Figure 3A); two pairs of wingpads present; tibiae of all legs shorter than femora (Figure 3B–D); gills I single, vestigial (Figure 4D); gills II double, operculate, jointed together medially; operculate plate quadrate, with dorsal ridge, ventral lamellae fimbriate (Figure 4B,C); gills III–V with double lamellae, fringed, dorsal lamella sub-oval, much larger than ventral lamella (Figure 4E–G); gills VI single, fimbriate (Figure 4H); posterolateral projections of terga V–IX well developed (Figure 5B); caudal filaments shorter than body, with ring of spine-like setae at articulations only (Figure 5C). Male imago: body length greater than 16.0 mm, wings with large yellowish brown to reddish markings (Figure 6B); forelegs with two blunt claws, mid- and hindlegs with one sharp and one blunt claw (Figure 7F–H); basal C-Sc crossveins of forewings well-developed (Figure 8); both forewings and hindwings with relatively numerous crossveins; MA of forewing forked at middle point, MP forked at base; hindwings with round costal projection, MA unforked, MP and Rs forked near middle; forceps 4-segmented, basal segment half- length of second segment, segmentation of apical segments incomplete; penes fused with small median cleft, penis lobes with clearly thickened lateral margins; three caudal filaments subequal in length and well developed. Female imago: body greater than 16.0 mm in length, both forewings and hindwing with markings as in male; hindwings with rounded costal projection as in male; subgenital plate and anal plate with straight posterior margins (Figure 9D). Subimago: all tarsal segments of both male and female subimagoes only with mi- crotrichia (Figure 10). Egg: long oval, with finger-like projections on surface (Figure 11G). Diagnosis: (see Table1) nymph with obvious frontal and genal projections, distinct anterolateral projections on pronotum, legs relatively short (tibiae shorter than femora), caudal filaments without swimming setae. Adults with colorful wings, rounded costal projection of hindwings, 4-segmented forceps and well developed median caudal filament.

Table 1. Comparison of characteristics of ﬁve genera within Neoephemeridae.

Genera Neoephemera Leucorhoenanthus Pulchephemera Potamanthellus Ochernova [1,5,9] Characters [1,3,5,18] [1,5,6,19,20] gen. n. [1,5] Frontal projection on head Absent Absent Absent Present Absent

Greatly or Without or Somewhat Without distinct Somewhat Mesonotal anterolateral expansions somewhat somewhat expanded expansions expanded expanded expanded

Maxillary palpi (length ratio of terminal 0.7–1.3 0.35 0.78–0.84 1.0–1.1 1.1–2.0 segment/2nd segment)

Labial palpi (length ratio of terminal 0.6–1.1 0.39 0.65 0.65 1.0–1.5 segment/2nd segment) Nymph Patella-tibial suture of mid- and hindlegs Present Absent Present Present Present

Longer than Shorter than Longer than Longer than Longer than Length of mid- and hind femora tibiae tibiae tibiae tibiae tibiae

Well developed Median tubercles on thoracic terga Absent Absent Absent Absent (except N. eatoni)

Without Without Without Without With swimming swimming setae; swimming setae; swimming setae; swimming setae; setae; ca. Caudal ﬁlaments ca. 0.8–1.0× ca. 1.4× length ca. 0.7× length ca. 0.45–0.5× 0.4–0.7× length length of body of body of body length of body of body Insects 2021, 12, 723 4 of 16

Table 1. Cont.

Genera Neoephemera Leucorhoenanthus Pulchephemera Potamanthellus Ochernova [1,5,9] Characters [1,3,5,18] [1,5,6,19,20] gen. n. [1,5] Ratio of distance between compound 0.15–0.5 / 1.15 0.1 0.04–0.15 eyes/diameter of compound eyes

One sharp, one Claws of forelegs / Two blunt Two blunt Two blunt blunt

Forewing length (mm) 8–17 9 8–11 18–20 6–10

Without Without Without with distinct with distinct Coloration of wings markings markings markings markings markings

Male Basal C-Sc crossveins of forewings Reduced Reduced Reduced Not reduced Not reduced imago Shape of basal costal projection of Acute Acute Acute Rounded Rounded hindwings

Well developed, Well developed, Well developed, Well developed, Vestigial, Forceps 4-segmented 4-segmented 4-segmented 4-segmented 3-segmented

Median incision of penis Small Small Small Small Wide

Median caudal ﬁlament Well developed Well developed Vestigial Well developed Vestigial

Length ratio of cerci/body 1.0–1.5 / 2.4 2.3–2.5 2.2–4.5

Remarks. Compared to the Eastern Asian Potamanthellus, this new genus has several different characters both in nymphal and imaginal stages: in nymphal stage, Pulchephemera has no swimming setae on caudal filaments and the projections of pronotum and mesonotum are distinct; in imaginal stage, the genitalia and terminal filament are well-developed. In contrast to the central Asian genus Ochernova, this new one possesses shorter legs (tibiae shorter than femora) and caudal filaments (ca 0.45–0.5× length of body) in nymphal stage. In imaginal stage, the coloration pattern of wings and the costal projection of hindwing of these two genera are totally different (see Table1). In the genus Leucorhoenanthus, the projections of pronotum and mesonotum are in- conspicuous which are well-developed in our new genus. In addition, the adults of Leucorhoenanthus also have the colorless wings and vestigial terminal filament (see Table1). The American genus Neoephemera possesses unique characters in both nymphal and imaginal stages: tubercles always present on the nota of nymph (absent in all other genera including Pulchephemera) and the foreleg of male imago have one sharp and one blunt claw (which both blunt in Pulchephemera, Leucorhoenanthu and Potamanthellus). This new genus has some combined characters of two groups in this family: the colorful wings and rounded costal projection of hindwings are similar to Potamanthellus; the 4-segmented forceps, fused penes and the absence of swimming setae in nymphal caudal filaments are alike to Neoephemera-like group. At the same time, the adults of this new genus have two unique characters and plesiomorphies at least: more crossveins on wings, MP and Rs of hindwings forked in the middle. The nymphs of the new genus have two characters of their own too: extended frons and genal projections. The large body of this new genus are also contributive to their identification. The forewing Cu area (between CuA and CuP) of the new genus is larger than that of Neoephemera but smaller than Potamanthellus. Eggs are slightly longer and with scattered finger-like projections on surface than those known eggs in the family. Etymology: the genus name Pulchephemera is derived from Latin “pulch-” and “ephemera” which means beauty or beautiful mayfly and refers to the colorful wings of the adults.

3.1. Description Pulchephemera projecta (Zhou and Zheng, 2001) comb. n. Neoephemera projecta Zhou and Zheng, 2001: 328, Figures1–9, nymph. Type: nymph, from Yunnan and Sichuan, China. Insects 2021, 12, x FOR PEER REVIEW 5 of 17

genus have two unique characters and plesiomorphies at least: more crossveins on wings, MP and Rs of hindwings forked in the middle. The nymphs of the new genus have two characters of their own too: extended frons and genal projections. The large body of this new genus are also contributive to their identification. The forewing Cu area (between CuA and CuP) of the new genus is larger than that of Neoephemera but smaller than Pota- manthellus. Eggs are slightly longer and with scattered finger-like projections on surface than those known eggs in the family. Etymology: the genus name Pulchephemera is derived from Latin “pulch-” and “ephemera” which means beauty or beautiful mayfly and refers to the colorful wings of the adults.

Insects 2021, 12, 723 3.1. Description 5 of 16 Pulchephemera projecta (Zhou and Zheng, 2001) comb. n. Neoephemera projecta Zhou and Zheng, 2001: 328, Figures 1–9, nymph. Type: nymph, from Yunnan and Sichuan, China. Neoephemera projecta: Kluge, 2004: 276; Bauernfeind and Soldán, 2012: 514; Holland Neoephemera projecta: Kluge, 2004: 276; Bauernfeind and Soldán, 2012: 514; Holland et etal., al., 2016: 2016: 140. 140.

FigureFigure 1. Nymphal habitus habitus of of PulchephemeraPulchephemera projecta projecta: (A:()A dorsal) dorsal view view (immature (immature nymph); nymph); (B) ventral (B) ventral view (immature nymph); (C) dorsal view (mature nymph); (D) ventral view (mature nymph). view (immature nymph); (C) dorsal view (mature nymph); (D) ventral view (mature nymph).

MaterialMaterial examined. Mature Mature nymph nymph (Holotyp (Holotype),e), Chuan-Zhu-Shi Chuan-Zhu-Shi (32.39° (32.39 N,◦ 103.35°N, 103.35 E),◦ E), Songpan County, Sichuan Province, 18-viii-2000, ZHOU Chang-fa. 3 nymphs (Paratypes); Songpan County, Sichuan Province, 18-viii-2000, ZHOU Chang-fa. 3 nymphs (Paratypes); same data as holotype; 8 nymphs, Chuan-Zhu-Shi, Songpan County, Sichuan Province, same data as holotype; 8 nymphs, Chuan-Zhu-Shi, Songpan County, Sichuan Province, 11-viii-2000, ZHOU Chang-fa; 12 male imagoes, 15 female imagoes, 2 male subimagoes, 1 11-viii-2000, ZHOU Chang-fa; 12 male imagoes, 15 female imagoes, 2 male subimagoes, female subimagoes, 25 nymphs, Potatso National Park, Shangri-La City, Yunnan Prov- 1 female subimagoes, 25 nymphs, Potatso National Park, Shangri-La City, Yunnan Province, ince, 1–25-iv-2021, MA Zhen-xing and MU Pen-xu; 5 nymphs, Qinggangshu Village, Xi’an 1–25-iv-2021, MA Zhen-xing and MU Pen-xu; 5 nymphs, Qinggangshu Village, Xi’an City, City, Shaanxi Province, 7-viii-2019, HAN Na and ZHANG Ming. Material deposited in Shaanxi Province, 7-viii-2019, HAN Na and ZHANG Ming. Material deposited in the the Department of Biology, Nanjing Normal University, Nanjing, China. DepartmentNymph: of body Biology, length Nanjing 19.0–22.0 Normal mm (mature), University, caudal Nanjing, filaments China. length 8.0–10.0 (0.45– 0.5× Nymph:body), body body yellowish length brown 19.0–22.0 (immature) mm (mature),to brownish caudal black (mature) filaments (Figure length 1). 8.0–10.0 (0.45–0.5Head:× generallybody), body brownish yellowish black. brown Antennae (immature) with dark to brownish base, other black portion (mature) pale (orFigure col- 1 ). orless,Head: slightly generally longer than brownish head width, black. arti Antennaeculations withwith tiny dark setae; base, frons other expanded portion and pale or colorless,forming a slightly distinct longer projection, than anterior head width, margin articulations slightly to with moderately tiny setae; concave frons expanded(obviously and formingconcave in a distinctspecimens projection, of Shaanxi anterior but nearly margin straight slightly in specimens to moderately of Yunnan); concave Genae (obviously en- concavelarged into in specimensclear projections of Shaanxi (Figure but 2A). nearly Dorsal straight surface inof head specimens with scattered of Yunnan); furcate- Genae enlargedstout setae, into margins clear projectionsof frontal projection (Figure2 andA). Dorsalgenal projections surface of with head more with such scattered setae (Fig- furcate- stoutures 2A setae, and margins11A); head of vertex frontal coarse projection (Figure and 2A). genalLabrum: projections anterior margin with more slightly such con- setae (caveFigure medially2A and (Figure Figure 2B), 11A); dorsal head surface vertex densely coarse covered (Figure with2A). setae Labrum: which anteriorlook like marginlong slightlyplumose, concave stout plumose medially or (Figure bifurcate2B), (Figure dorsal surface11B); ventral densely surface covered densely with setaecovered which with look like long plumose, stout plumose or bifurcate (Figure 11B); ventral surface densely covered with long and hair-like plumose setae. Mandibles: outer margin and dorsal surface densely covered with bifurcate setae (Figure 11C); outer incisor with 3–4 teeth, inner incisor with 2–3 teeth; prostheca divided into a tuft of fine setae and a single tooth; inner margin near mola with a row of dense fine setae (Figure2C,D). Hypopharynx: anterior margin of lingua slightly concave medially, covered with dense fine setae; superlinguae with long hair-like setae apically (Figure2E). Maxilla: galea-lacinia with 3 canines and 2 dentisetae, both inner and outer margins with row of long hair-like plumose setae; three segments of maxillary palp subequal in length, segment I distinct broader than segments II and III, with bifurcate- plumose setae along inner and outer margins, segment II with long hair-like setae, segment III with setae on distal half (Figure2F). Labium: glossae with round pointed apex and subapical stout spine-like setae, ventral surface with dense long hair-like setae; paraglossae with dense long hair-like setae on both dorsal and ventral surface; segment I of labial palp expanded, segment II broader than segment III; length of segment I subequal to segment II, segment III ca. 2/3 length of segment II; segment I with dense bifurcate-plumose setae on ventral surface, segment II with stout bifurcate-plumose setae on ventral surface and long hair-like setae near outer margin; segment III with 7–10 subapical stout spine-like setae Insects 2021, 12, x FOR PEER REVIEW 6 of 17

long and hair-like plumose setae. Mandibles: outer margin and dorsal surface densely covered with bifurcate setae (Figure 11C); outer incisor with 3–4 teeth, inner incisor with 2–3 teeth; prostheca divided into a tuft of fine setae and a single tooth; inner margin near mola with a row of dense fine setae (Figure 2C,D). Hypopharynx: anterior margin of lingua slightly concave medially, covered with dense fine setae; superlinguae with long hair- like setae apically (Figure 2E). Maxilla: galea-lacinia with 3 canines and 2 dentisetae, both inner and outer margins with row of long hair-like plumose setae; three segments of maxillary palp subequal in length, segment I distinct broader than segments II and III, with bifurcate-plumose setae along inner and outer margins, segment II with long hair-like setae, segment III with setae on distal half (Figure 2F). Labium: glossae with round pointed apex and subapical stout spine-like setae, ventral surface with dense long hair-like setae; paraglossae with dense long hair-like setae on both dorsal and ventral surface; segment I Insects 2021, 12, 723 of labial palp expanded, segment II broader than segment III; length of segment I subequal6 of 16 to segment II, segment III ca. 2/3 length of segment II; segment I with dense bifurcate- plumose setae on ventral surface, segment II with stout bifurcate-plumose setae on ventral surface and long hair-like setae near outer margin; segment III with 7–10 subapical stout andspine-like covered setae with and dense covered long hair-likewith dense setae long near hair-like outer margin; setae near mentum outer andmargin; submentum mentum withand bifurcate-plumosesubmentum with bifurcate-plumose setae on ventral surface setae on (Figure ventral2G). surface (Figure 2G).

FigureFigure 2. 2.Head Head and and mouthparts mouthparts ofofPulchephemera Pulchephemeraprojecta projecta:(: (AA)) head;head; (B(B)) labrum; labrum; (C(C)) left left mandible; mandible; (D) right mandible; (E) hypopharynx; (F) maxillae; (G) labium. (D) right mandible; (E) hypopharynx; (F) maxillae; (G) labium.

Thorax:Thorax: brownbrown (immature) (immature) to to brownish brownish black black (mature); (mature); pronotum pronotum expanded expanded laterally, later- withally, distinctwith distinct anterolateral anterolateral projections; projections; anterolateral anterolateral projections projections of mesonotum of mesonotum extended ex- to sametended level to same of pronotum level of pron (Figureotum3A). (Figure Margins 3A). of Margins thorax with of thorax dense with stout dense bifurcate stout setae, bifur- dorsalcate setae, and ventraldorsal and surface ventral of thorax surface covered of thor withax covered scattered with stout scattered bifurcate stout setae. bifurcate setae. Legs: fore femora with a transverse row of bifurcate setae subapically (Figure 11D); femoraLegs: of mid- fore and femora hindlegs with similar,a transverse with scatteredrow of bifurcate stout bifurcate setae subapically setae on both (Figure dorsal 11D); and ventralfemora surface; of mid- ventral and hindlegs surface similar, of tibiae with with scattered scattered stout bifurcate setae;setae inneron both margins dorsal of both tibiae and tarsi with long plumose setae and ﬁne setae. Claws of all legs with acute apex (Figure3B–D). Length ratio of foreleg femur: tibia: tarsus = 1.9: 1.1: 1.5; midleg femur: tibia: tarsus = 2.1: 1.3: 1.3; hindleg femur: tibia: tarsus = 2.9: 1.7: 1.5. Patella-tibial suture of mid- and hindlegs clear expressed. Coxae and trochanter with setae too (Figure3B–D). Insects 2021, 12, x FOR PEER REVIEW 7 of 17

and ventral surface; ventral surface of tibiae with scattered stout bifurcate setae; inner margins of both tibiae and tarsi with long plumose setae and fine setae. Claws of all legs with acute apex (Figure 3B–D). Length ratio of foreleg femur: tibia: tarsus = 1.9: 1.1: 1.5; Insects 2021, 12, 723 midleg femur: tibia: tarsus = 2.1: 1.3: 1.3; hindleg femur: tibia: tarsus = 2.9: 1.7: 1.5. Patella-7 of 16 tibial suture of mid- and hindlegs clear expressed. Coxae and trochanter with setae too (Figure 3B–D).

FigureFigure 3. ThoracicThoracic structures structures of ofPulchephemeraPulchephemera projecta projecta: (A):( thorax;A) thorax; (B) foreleg; (B) foreleg; (C) middle (C) middleleg; (D) leg; hind leg. (D) hind leg. Abdomen: brown to brownish black; terga I–II and VI–X with bifurcate setae along Abdomen: brown to brownish black; terga I–II and VI–X with bifurcate setae along posterior margins and median line of each tergum (Figures 5A and 11F); sterna covered posterior margins and median line of each tergum (Figure5A and Figure 11F); sterna with dense stout bifurcate setae. Terga I–II with distinct posteromedial tubercle (Figure covered with dense stout bifurcate setae. Terga I–II with distinct posteromedial tubercle 4A); segments V–IX with well-developed posterolateral projections, progressively larger (Figure4A); segments V–IX with well-developed posterolateral projections, progressively posteriorly (Figure 5B). Gills I–VI present; gill I single, with 2 segments, apical one obvi- larger posteriorly (Figure5B). Gills I–VI present; gill I single, with 2 segments, apical ously longer than basal one and covered with long plumose setae and scattered fine setae one obviously longer than basal one and covered with long plumose setae and scattered (Figure 4D); gills II with dorsal subquadrate operculate plate meeting medially (Figure fine4A,B), setae dorsal (Figure diagonal4D); gillsridge II on with each dorsal opercu subquadratelum distinct operculateand covered plate with meetingscattered medially stout (Figurebifurcate4A,B), setae dorsal (Figure diagonal 11E), free ridge margins on each with operculum long plumose distinct setae and and covered fine setae; with ventral scattered stoutlamella bifurcate much smaller setae (Figure than dorsal 11E), operculum, free margins divided with longinto several plumose long setae fringes and (Figure fine setae; ventral4C). Gill lamella III–V similar much in smaller shape and than structure, dorsal operculum, dorsal lamell dividedae “kidney-shaped” into several longand with fringes (Figurerow of4 fringesC). Gill on III–V medial similar margin in shape and andlateral structure, margin, dorsal ventral lamellae lamellae “kidney-shaped” similar to gill II and with row of fringes on medial margin and lateral margin, ventral lamellae similar to gill II counterparts (Figure4E–G); Gill VI single, similar to those dorsal lamellae of gills III–V (Figure4H). Three caudal filaments subequal in length, with whorls of spine-like setae between articulations (Figure5C). Insects 2021, 12, x FOR PEER REVIEW 8 of 17

Insects 2021, 12, x FOR PEER REVIEW 8 of 17

Insects 2021, 12, 723 counterparts (Figure 4E–G); Gill VI single, similar to those dorsal lamellae of gills III–V8 of 16 counterparts(Figure 4H). Three(Figure caudal 4E–G); filaments Gill VI single, subequal similar in length, to those with dorsal whorls lamellae of spine-like of gills III–Vsetae (Figurebetween 4H). articulations Three caudal (Figure filaments 5C). subequal in length, with whorls of spine-like setae between articulations (Figure 5C).

FigureFigure 4.4. AbdominalAbdominal structures of of PulchephemeraPulchephemera projecta projecta: (A:()A abdominal) abdominal terga terga I–VI; I–VI; (B) (operculateB) operculate Figuregill II (dorsal 4. Abdominal view); ( Cstructures) operculate of Pulchephemera gill II (ventral projecta view); :( D(A)) gill abdominal I; (E) gill terga III; ( FI–VI;) gill (IV;B) operculate(G) gill V; gill II (dorsal view); (C) operculate gill II (ventral view); (D) gill I; (E) gill III; (F) gill IV; (G) gill V; gill(H) IIgill (dorsal VI. Abbreviations: view); (C) operculate dl, dorsal gill lamella; II (ventral vl, ventral view); lamella; (D) gill pt,I; ( Eposterior) gill III; tubercle.(F) gill IV; (G) gill V; (H(H)) gillgill VI.VI. Abbreviations:Abbreviations: dl, dl, dorsal dorsal lamella; lamella; vl, vl, ventral ventral lamella; lamella; pt, pt, posterior posterior tubercle. tubercle.

Figure 5. Abdominal structures of Pulchephemera projecta: (A) abdominal terga VI–X; (B) abdominal FigureFiguresterna; 5.5.(C Abdominal) caudal filaments. structures structures of of PulchephemeraPulchephemera projecta projecta: (A:()A abdominal) abdominal terga terga VI–X; VI–X; (B) (abdominalB) abdominal sterna; (C) caudal filaments. sterna; (C) caudal ﬁlaments. Male imago: body length 17.0–20.0 mm, forewing 18.0–20.0 mm, hindwing 7.5–8.0 mm,Male Malecaudal imago:imago: filaments bodybody 40.0–45.0 lengthlength 17.0–20.017.0–20.0 mm (2.3–2.5× mm,mm, forewingforewing length of 18.0–20.0 18.0–20.0 body), body mm, mm, hindwing reddishhindwing brown 7.5–8.0 7.5–8.0 to mm, caudalmm,brownish caudal ﬁlaments black filaments (Figure 40.0–45.0 40.0–45.0 6B). mm (2.3–2.5 mm (2.3–2.5×× length length of body), of body), body reddishbody reddish brown brown to brownish to brownish black (Figure 6B). blackHead: (Figure Compound6B). eye: upper portion reddish grey, basal portion grey (eyes brown Head: Compound eye: upper portion reddish grey, basal portion grey (eyes brown to darkHead: in living) Compound (Figures eye: 6B upperand 7A); portion distance reddish between grey, eyes basal subequal portion to greywidth (eyes of median brown to toocellus dark (aboutin living) 0.10× (Figures dorsal 6B diameter and 7A); of distance one compound between eye) eyes (Figure subequal 7A). to Frontal width ofprojection median dark in living) (Figures6B and7A); distance between eyes subequal to width of median ocelluswith straight (about anterior 0.10× dorsal margin. diameter Vertex of coarse one co (Figurempound 7A). eye) (Figure 7A). Frontal projection ocellus (about 0.10× dorsal diameter of one compound eye) (Figure7A). Frontal projection with Thorax:straight unicolorousanterior margin. reddish Vertex brown coarse to (Figuredark brown, 7A). with a weak sutural ommation with straight anterior margin. Vertex coarse (Figure7A). (FigureThorax: 7A); furcasternalunicolorous protuberancesreddish brown dark to dark brown, brown, separated with a widely weak sutural (Figure ommation 7B). Fore- Thorax: unicolorous reddish brown to dark brown, with a weak sutural ommation (Figurelegs darker 7A); thanfurcasternal mid- and protuberances hindlegs; femora dark brown,of all legs separated darker widelythan tibiae (Figure or tarsi,7B). Fore-with (Figurelegs darker7A); furcasternalthan mid- and protuberances hindlegs; femora dark brown,of all legs separated darker widelythan tibiae (Figure or 7tarsi,B). Forelegs with darker than mid- and hindlegs; femora of all legs darker than tibiae or tarsi, with basal and apical dark dots (indistinct in mid- and hind femora) (Figure7C–E). Ratios of fore femora: tibiae: tarsi = 2.9: 4.6: 5.4, ratios of fore tarsal segments I: II: III: IV: V = 0.2: 1.8: 1.5: 1.1: 0.7; Ratios of middle femora: tibiae: tarsi =2.5: 1.9: 1.8; ratios of hind femora: tibiae: tarsi = 3.5: 2.5: 1.9. First tarsal segment of mid- and hindlegs shorter than others, Insects 2021, 12, x FOR PEER REVIEW 9 of 17

Insects 2021, 12, x FOR PEER REVIEW 9 of 17

Insects 2021, 12, 723 basal and apical dark dots (indistinct in mid- and hind femora) (Figure 7C–E). Ratios of9 of 16 basalfore femora: and apical tibiae: dark tarsi dots = 2.9: (indistinct 4.6: 5.4, ratiosin mid- of foreand tarsalhind femora)segments (Figure I: II: III: 7C–E). IV: V Ratios= 0.2: 1.8: of fore1.5: 1.1:femora: 0.7; Ratios tibiae: of tarsi middle = 2.9: femora: 4.6: 5.4, tibiae: ratios tarsiof fore =2.5: tarsal 1.9: segments1.8; ratios I: of II: hind III: IV:femora: V = 0.2: tibiae: 1.8: 1.5:tarsi 1.1: = 3.5: 0.7; 2.5: Ratios 1.9. ofFirst middle tarsal femora: segment tibiae: of mid- tarsi and =2.5: hindlegs 1.9: 1.8; shorter ratios of than hind others, femora: fifth tibiae: seg- ﬁfth segment longest, segments 2–4 subequal in length. Foreleg with two blunt claws; mid- tarsiment = longest, 3.5: 2.5: segments1.9. First tarsal 2–4 subequal segment inof mid-length. and Foreleg hindlegs with shorter two blunt than others,claws; mid-fifth seg-and and hindlegs with one acute and one blunt claw (Figure7F–H). menthindlegs longest, with segmentsone acute 2–4and subequalone blunt in claw leng (Figureth. Foreleg 7F–H). with two blunt claws; mid- and hindlegs with one acute and one blunt claw (Figure 7F–H).

Figure 6. Adult habitus of Pulchephemera projecta: (A) male subimago; (B) male imago; (C) female Figure 6. Adult habitus of Pulchephemera projecta:(A) male subimago; (B) male imago; (C) female Figuresubimago; 6. Adult (D) female habitus imago. of Pulchephemera projecta: (A) male subimago; (B) male imago; (C) female subimago;subimago; ((D) female imago. imago.

Figure 7. Male imago of Pulchephemera projecta: (A) head and thorax (dorsal view); (B) head and FigureFigurethorax 7.(ventral7. Male imagoview); (of ofC )Pulchephemera Pulchephemeraforeleg; (D) middle projecta projecta leg;: (:(A ()EA )head) hindleg; head and and (thoraxF) thoraxclaw (dorsal (foreleg); (dorsal view); view);(G) (clawB) (headB (middle) head and and thoraxleg); (H (ventral) claw (hindleg). view); (C ) foreleg; (D) middle leg; (E) hindleg; (F) claw (foreleg); (G) claw (middle thorax (ventral view); (C) foreleg; (D) middle leg; (E) hindleg; (F) claw (foreleg); (G) claw (middle leg); leg); (H) claw (hindleg). (H) claw (hindleg). Wings: almost entire forewings and hindwings covered with purplish to brown markingsWings: except almost Cu entireto A fields forewings (Figure and 8); bothhindwings forewings covered and hind withwings purplish with torelatively brown Wings: almost entire forewings and hindwings covered with purplish to brown markingsdense crossveins except Cuincluding to A fields basal (Figure portion 8); of both C and forewings Sc. Forewings: and hind Rswings forked with at relativelymidpoint markingsdense crossveins except Cuincluding to A ﬁelds basal (Figure portion8 );of both C and forewings Sc. Forewings: and hindwings Rs forked withat midpoint relatively dense crossveins including basal portion of C and Sc. Forewings: Rs forked at midpoint between base of MA+Rs and fork of MA; MP forked near base; MP2, CuA and CuP recurved; CuP ends near midpoint of hind margin; A1 single, with two veinlets; bullae of Sc, Rs clear, crossveins of stigma oblique, some of them S-shaped. Hindwings ca. 0.4×

forewing length, with rounded costal projection at base; MA single, Rs fork at about same level as MP fork (Figure8). Insects 2021, 12, x FOR PEER REVIEW 10 of 17

Insects 2021, 12, x FOR PEER REVIEW 10 of 17

between base of MA+Rs and fork of MA; MP forked near base; MP2, CuA and CuP re- betweencurved; CuPbase endsof MA+Rs near midpointand fork of of MA; hind MP margin; forked A1 near single, base; withMP2, two CuA veinlets; and CuP bullae re- of curved;Sc, Rs clear, CuP endscrossveins near midpoint of stigma of hindoblique, margin; some A1 of single, them withS-shaped. two veinlets; Hindwings bullae ca. of 0.4x Insects 2021, 12, 723 Sc,forewing Rs clear, length, crossveins with roundedof stigma costal oblique, projection some of at them base; S-shaped. MA single, Hindwings Rs fork at ca.about 0.4x same 10 of 16 forewinglevel as MP length, fork with (Figure rounded 8). costal projection at base; MA single, Rs fork at about same level as MP fork (Figure 8).

Figure 8. Wings of male imago of Pulchephemera projecta. FigureFigure 8.8. Wings of male imago imago of of PulchephemeraPulchephemera projecta projecta. .

Figure 9. Abdominal structures of adults of Pulchephemera projecta: (A) abdominal terga of male imago; (B) abdominal sterna of male imago; (C) abdominal terga I–II (lateral view) of male; (D) FiguresternaFigure VII–IX 9.9. Abdominal of female structuresimago; structures (E) genitaliaof of adults adults (ventral of of PulchephemeraPulchephemera view); (F) penis projecta projecta lobes: ( (dorsalA:() Aabdominal)abdominal view). terga terga of male of male imago; (B) abdominal sterna of male imago; (C) abdominal terga I–II (lateral view) of male; (D) imago; (B) abdominal sterna of male imago; (C) abdominal terga I–II (lateral view) of male; (D) sterna sterna VII–IX of female imago; (E) genitalia (ventral view); (F) penis lobes (dorsal view). VII–IX of female imago; (E) genitalia (ventral view); (F) penis lobes (dorsal view).

Abdomen: reddish brown to dark brown (Figure 9A,B), terga I–II with posteromedial tubercles (reduced tubercles of nymph), that of tergum II much larger (Figure 9C); terga VIII–IX with clear posterolateral projections (Figure 9B), those of terga IX much longer; each sternum with a pair of short dark stripes and submedian paired dots, area near lateral margin darker than medial area (Figure 9B). Genitalia (Figure 9E,F): posterior margin of Insects 2021, 12, 723 styliger plate straight; forceps yellow, paler than terga or subgenital plate; segment I 11of of 16 forceps broader than others, with clear inner projection apically; segment II ca. 2.0× length of segment I, curved inwards; segments III and IV very short, segmented incompletely marginsand tapered. and thatPenes area fused thicker with than median others. notch; Three penes brown very caudal thin, sperm filaments ducts with along tiny lateral setae on surface,margins terminaland that filamentarea thicker slightly than shorterothers. Three than cercibrown (Figure caudal6B). filaments with tiny setae on surface, terminal filament slightly shorter than cerci (Figure 6B). Female imago: body length 18.0–20.0 mm, forewing 20.0–22.0 mm, hindwing Female imago: body length 18.0–20.0 mm, forewing 20.0–22.0 mm, hindwing 8.0–8.5 8.0–8.5 mm, caudal filament length 30.0–34.0 mm (1.5–1.7× length of body), coloration mm, caudal filament length 30.0–34.0 mm (1.5–1.7× length of body), coloration pattern pattern similar to male imago (Figure6D). Wings similar to male. All legs with similar col- similar to male imago (Figure 6D). Wings similar to male. All legs with similar coloration oration as male imago, length ratios of femora: tibiae: tarsi of forelegs = 3.0: 2.5: 2.6; length as male imago, length ratios of femora: tibiae: tarsi of forelegs = 3.0: 2.5: 2.6; length ratios ratios of femora: tibiae: tarsi of middle legs = 2.8: 2.3: 2.2; length ratios of femora: tibiae: of femora: tibiae: tarsi of middle legs = 2.8: 2.3: 2.2; length ratios of femora: tibiae: tarsi of tarsi of hindlegs = 3.9: 2.9: 2.2; all legs with one blunt and one sharp claw. Abdominal hindlegs = 3.9: 2.9: 2.2; all legs with one blunt and one sharp claw. Abdominal coloration colorationsimilar to male, similar subgenital to male, subgenitalplate slightly plate expanded slightly posteriorly, expanded posteriorly,posterior margin posterior of suba- margin ofnal subanal plate nearly plate straight nearly straight(Figure 9D). (Figure 9D). MaleMale subimago: body body length length 19.0 19.0 mm, mm, forewing forewing 19.0 19.0 mm, mm, hindwing hindwing 8.0 8.0mm, mm, caudal caudal filamentsfilaments 20 mm (ca 1.0× 1.0× lengthlength of of body); body); colo colorationration similar similar to male to male imago imago but butsomewhat somewhat fainterfainter (Figure(Figure 66A).A). Wings semi-hyaline and and with with tiny tiny setae setae along along margins. margins. Legs Legs darker darker thanthan thosethose of male imago, imago, length length of of tarsi tarsi in in forelegs forelegs subequal subequal to totibiae, tibiae, all alllegs legs with with one one bluntblunt andand one sharp claws; claws; all all tarsal tarsal segments segments of ofall all legs legs covered covered by bydense dense microtrichiae microtrichiae (Figure(Figure 1010A–F).A–F). Caudal filaments filaments with with dense dense se setaetae on on surface, surface, terminal terminal filament filament slightly slightly shortershorter than cerci.

FigureFigure 10. SubimaginalSubimaginal tarsi tarsi of Pulchephemera of Pulchephemera projecta projecta: (A) tarsi:(A of) tarsiforeleg of (male foreleg subimago); (male subimago); (B–C) tarsal segment 5 of foreleg and the details (male subimago) (D) base of tarsal segment 5 of foreleg (B,C) tarsal segment 5 of foreleg and the details (male subimago) (D) base of tarsal segment 5 of foreleg (male subimago); (E) details of tarsal segment 1 of foreleg (male subimago); (F) apex of tarsal segment 5 of midleg (male subimago); (G) tarsal segment 4 of hindleg (female subimago).

Female subimago: body length 20.0 mm, forewing 20.0 mm, hindwing 8.5 mm, caudal ﬁlament length 20 mm (ca 1.0× length of body), coloration similar to male subimago (Figure6C). All legs with one blunt and one sharp claws; same as male subimago, all tarsal segments covered by dense microtrichiae (Figure 10G). Eggs: length 210–240 µm; width 140–150 µm, oval (Figure 11G), ﬁnger-like projections (fp) 12–16 µm, scattered on the surface (Figure 11G,H), these projections consist of many thin threads and somewhat expanded apically (Figure 11H). Tagenoform micropyle located in the equatorial area, sperm guide round, diameter 17–20 µm (Figure 11G). Insects 2021, 12, x FOR PEER REVIEW 12 of 17

(male subimago); (E) details of tarsal segment 1 of foreleg (male subimago); (F) apex of tarsal segment 5 of midleg (male subimago); (G) tarsal segment 4 of hindleg (female subimago).

Female subimago: body length 20.0 mm, forewing 20.0 mm, hindwing 8.5 mm, caudal filament length 20 mm (ca 1.0× length of body), coloration similar to male subimago (Figure 6C). All legs with one blunt and one sharp claws; same as male subimago, all tarsal segments covered by dense microtrichiae (Figure 10G). Eggs: length 210–240 μm; width 140–150 μm, oval (Figure 11G), finger-like projec- Insects 2021, 12, 723 tions (fp) 12–16 μm, scattered on the surface (Figure 11G,H), these projections consist12 of 16of many thin threads and somewhat expanded apically (Figure 11H). Tagenoform micropyle located in the equatorial area, sperm guide round, diameter 17–20 μm (Figure 11G).

Figure 11. Setae of nymph and eggs of Pulchephemera projecta:(A) anterior margin of frontal projection; (B) dorsal view of labrum (left half); (C) lateral margin of mandible; (D) transverse row of setae on fore femora; (E) setae on diagonal ridge of gill II; (F) posterior margin of abdominal tergum VII;

(G) eggs; (H) detail view of surface of eggs. Abbreviations: m, micropyle; sbs, stout bifurcate setae; ps, plumose setae; bs, bifurcate setae; fp, ﬁnger-like projections.

3.2. Biology and Remarks Nymphs of this species (collected in 2021) live in small to middle sized streams (1.0–8.0 m wide, 0.1–1.5 m deep, ca. 2500–3400 m altitude, Figure 12A), and they are found underneath stones in moderate to fast ﬂowing sections where the substrate is stony, most stone covered with mud and submerged plants (Figure 12C). The nymphs move Insects 2021, 12, x FOR PEER REVIEW 13 of 17

Figure 11. Setae of nymph and eggs of Pulchephemera projecta: (A) anterior margin of frontal projection; (B) dorsal view of labrum (left half); (C) lateral margin of mandible; (D) transverse row of setae on fore femora; (E) setae on diagonal ridge of gill II; (F) posterior margin of abdominal tergum VII; (G) eggs; (H) detail view of surface of eggs. Abbreviations: m, micropyle; sbs, stout bifurcate setae; ps, plumose setae; bs, bifurcate setae; fp, finger-like projections.

3.2. Biology and Remarks Insects 2021, 12, 723 Nymphs of this species (collected in 2021) live in small to middle sized streams (1.0–13 of 16 8.0 m wide, 0.1–1.5 m deep, ca. 2500–3400 m altitude, Figure 12A), and they are found underneath stones in moderate to fast flowing sections where the substrate is stony, most stone covered with mud and submerged plants (Figure 12C). The nymphs move slowly slowly in the water, have weak swimming ability, their body are usually covered with in the water, have weak swimming ability, their body are usually covered with dense dense muddy debris (especial immature nymphs, as Figure 12B). muddy debris (especial immature nymphs, as Figure 12B).

FigureFigure 12.12.Habitat Habitat and and emergence emergence process process of ofPulchephemera Pulchephemera projecta projecta:(A: )(A habitat;) habitat; (B) immature(B) immature nymph nymph alive; (C) mature nymph alive; (D) subimago in the process of emergence; (E) subimago on alive; (C) mature nymph alive; (D) subimago in the process of emergence; (E) subimago on the water the water surface; (F) subimago on the bank of the river. surface; (F) subimago on the bank of the river. Observed emergence happened from early to the end of April. Subimagoes emerged Observed emergence happened from early to the end of April. Subimagoes emerged between 4:30 and 6:00 p.m. local time, and this process occurred at the surface of stream between 4:30 and 6:00 p.m. local time, and this process occurred at the surface of stream wa- water (nymphs float to the surface of the water and then become subimagoes, see Figure ter (nymphs float to the surface of the water and then become subimagoes, see Figure 12D, 12D, the molting nymph in the photo was not in its natural state but was placed on a thestone). molting After nymph that, the in subimagoes the photo was drift not downstream in its natural with state the but water was placedcurrent onon a its stone). surfaceAfter that, the subimagoes drift downstream with the water current on its surface (Figure 12E) and then climb onto stones or stream banks when they meet them (Figure 12F). Life span of subimagoes in the laboratory ranges from 53–68 h. Molting of the subimago to imago was observed both in evening and daytime, the process lasts ca. 14–17 min. Distribution. China (Shaanxi, Yunnan and Sichuan). Egg diagnosis. The eggs of this species are more similar to the species of the Neoephemera- group because of their finger-like projections [1]. Compared to eggs of Neoephemera youngi, N. eatoni and L. maximus [3,21], the finger-like projections of Pulchephemera projecta are stouter and irregularly situated. Insects 2021, 12, x FOR PEER REVIEW 14 of 17

(Figure 12E) and then climb onto stones or stream banks when they meet them (Figure 12F). Life span of subimagoes in the laboratory ranges from 53–68 h. Molting of the subimago to imago was observed both in evening and daytime, the process lasts ca. 14–17 min. Distribution. China (Shaanxi, Yunnan and Sichuan). Egg diagnosis. The eggs of this species are more similar to the species of the Ne- Insects 2021, 12, 723 oephemera-group because of their finger-like projections [1]. Compared to eggs of Ne- 14 of 16 oephemera youngi, N. eatoni and L. maximus [3,21], the finger-like projections of Pulchephemera projecta are stouter and irregularly situated.

4. Discussion4. Discussion ConsideringConsidering a series a series of plesiomorphies of plesiomorphies of P. projecta of P. projecta(such as (suchlarge body as large and bodywing and wing size,size, more more crossveins, crossveins, well-devel well-developedoped genitalia genitalia and terminal and filament), terminal we filament), regard it as we the regard it as mostthe primitive most primitive taxon in Neoephemeridae. taxon in Neoephemeridae. Therefore, the characters Therefore, of its the wings characters (with dark of its wings markings(with dark and hindwings markings with and blunt hindwings costal proj withection) blunt are costal also considered projection) as the are primitive also considered as statethe of primitive this family state rather of than this th familye synapomorphies rather than of the the synapomorphies new genus and Potamanthellus of the new. genus and For the same reason, the frontal projection in nymphal stage is also regarded as a plesio- Potamanthellus. For the same reason, the frontal projection in nymphal stage is also regarded morphy of this species and the family as well. as a plesiomorphy of this species and the family as well. Historically, the phylogenetic position of the family Neoephemeridae has been changedHistorically, several times the [1,5,10,11,22–26]. phylogenetic positionIn the system of the of family Kluge, Neoephemeridae the relationship between has been changed Potamantus/fg2,several times [Euthyplocia/1,5,10,11,22fg1,–26 ].Fossoriae In the system and Caenotergaliae of Kluge, the relationship(=Neoephemera/fg1+ between Potaman- Caenoptera)tus/fg2, Euthyplocia/fg1, is unclear. Based on Fossoriae the characters and Caenotergaliae of P. projecta described (=Neoephemera/fg1+ in this work, we Caenoptera) proposeis unclear. two hypotheses Based on on the this characters issue (Figure of P.13 projecta). The firstdescribed one is that in the this nymphal work, frontal we propose two projectionhypotheses is the on synapomorphy this issue (Figure of Fimbriatot 13). Theergaliae, first one which is that then the were nymphal secondarily frontal lost projection is in thePotamantus/fg2, synapomorphy Euthyplocia/fg1 of Fimbriatotergaliae, and Caenoptera. which The then second were one secondarily hypothesizes lost that in Potaman- thetus/fg2, frontal projection Euthyplocia/fg1 is the synapomorphy and Caenoptera. of Caenotergaliae+Fossori The second oneae hypothesizes only. Meanwhile, that the frontal theprojection dorsal-oriented is the gills synapomorphy may also be anothe of Caenotergaliae+Fossoriaer synapomorphy of Caenotergaliae+Fossoriae. only. Meanwhile, the dorsal- Similaroriented frontal gills projection may also found be anotherin some mayfly synapomorphy taxa which ofare Caenotergaliae+Fossoriae. not belonging to Fimbria- Similar totergaliae, such as Drunella/g1 [5], is obvious homoplasy and the imaginal characters of frontal projection found in some mayfly taxa which are not belonging to Fimbriatotergaliae, them were totally different. such as Drunella/g1 [5], is obvious homoplasy and the imaginal characters of them were totally different.

Figure 13. The two hypotheses of phylogeny of Fimbriatotergaliae: (A) hypothesis 1: frontal projec- Figure 13. The two hypotheses of phylogeny of Fimbriatotergaliae: (A) hypothesis 1: frontal projection of nymph is the synapomorphy of Fimbriatotergaliae; (B) hypothesis 2: frontal projection of nymphtion is of the nymph synapomorphy is the synapomorphy of Caenotergaliae+Fossoriae. of Fimbriatotergaliae; (B) hypothesis 2: frontal projection of nymph is the synapomorphy of Caenotergaliae+Fossoriae.

On the generic level, based upon phylogeny provided by Bae and McCafferty [1], we suggest a new hypothesis for our new genus (Figure 14). In this arrangement, the genus Pulchephemera was regarded as the ﬁrst divergent taxon of Neoephemeridae. The rest genera are grouped by the absence of frontal projection. Among them, the Neoephemera-like group left the Potamanthellus because they lost paintings on wings and have acute costal projection on hindwings. Insects 2021, 12, x FOR PEER REVIEW 15 of 17

On the generic level, based upon phylogeny provided by Bae and McCafferty [1], we suggest a new hypothesis for our new genus (Figure 14). In this arrangement, the genus Pulchephemera was regarded as the first divergent taxon of Neoephemeridae. The rest genera are grouped by the absence of frontal projection. Among them, the Neoephemera-like group left the Potamanthellus because they lost paintings on wings and have acute costal Insects 2021, 12, 723 15 of 16 projection on hindwings.

Figure 14. The hypothesis of the phylogenetic relation between the genera within Neoephemeridae Figure 14. The hypothesis of the phylogenetic relation between the genera within Neoephemeridae (partly(partly basedbased on on Bae Bae and and McCafferty McCafferty [1]). [1 ]).

Geographically,Geographically, we we believe believe that that the thefamily family Neoephemeridae Neoephemeridae originated originated from Eastern from Eastern AsiaAsia sincesince our new primitive primitive genus genus is isin inthis this region, region, then then dispersed dispersed western-wards. western-wards. Dur- During thising this process, process, the the other other genera genera split split and and originatedoriginated progressively. progressively. The The most most plesiomor- plesiomorphic mayflyphic mayflySiphluriscus Siphluriscus chinensis chinensisUlmer, Ulmer, 1920 is is distributed distributed in the in Orient the Orient too [27], too and [27 this], and this regionregion also has has the the most most diverse diverse species species of the of families the families Ephemerida Ephemeridaee and Potamanthidae. and Potamanthidae. SoSo far,far, severalseveral studies studies of of genus genus or orfamily-level family-level phylogeny phylogeny for mayfly for mayfly based on based molecular on molecular datadata havehave been published, and and they they either either verified verified the the traditional traditional morphological morphological results results or proposedor proposed new new hypotheses hypotheses [ 28[28–30].–30]. Therefor Therefore,e, analyses analyses for for phylogenies phylogenies of Neoephemer- of Neoephemeridae idae based on molecular evidence are necessary to test our theory in future work. based on molecular evidence are necessary to test our theory in future work. Author Contributions: Conceptualization, all authors; methodology, all authors; software, Z.M.; Authorvalidation, Contributions: all authors; formalConceptualization, analysis, all authors; all authors;investigation, methodology, all authors; allresources, authors; C.Z.; software, data Z.M.; validation,curation, Z.M.; all authors;writing—original formal analysis,draft preparation, all authors; Z.M.; investigation, writing—review all and authors; editing, resources, C.Z.; visu- C.Z.; data curation,alization, Z.M.;Z.M.; writing—originalsupervision, C.Z.; project draft preparation,administration, Z.M.; C.Z.; writing—review funding acquisition, and C.Z. editing, Both C.Z.;au- visual- ization,thors have Z.M.; read supervision, and agreed to C.Z.; the projectpublished administration, version of the manuscript. C.Z.; funding All acquisition,authors have C.Z. read Bothand authors haveagreed read to the and published agreed to version the published of the manuscript. version of the manuscript. All authors have read and agreed toFunding: the published This work version was funded of the by manuscript. the National Natural Science Foundation of China, grant number 31750002 and 32070475, the Priority Academic Program Development of Jiangsu Higher Educa- Funding:tion InstitutionsThis (PAPD), work was key fundedprojects of by science-technology the National Natural basic condition Science platform Foundation from The of Min- China, grant numberistry of Science 31750002 andand Technology 32070475, of the the People’s Priority Republic Academic of China, Program grant Development number 2005DKA21402, of Jiangsu Higher Educationand National Institutions Specimen (PAPD),Information key Infras projectstructure of science-technology (NSII), grant number basic 2005DKA21400. conditionplatform from The Ministry of Science and Technology of the People’s Republic of China, grant number 2005DKA21402, and National Specimen Information Infrastructure (NSII), grant number 2005DKA21400. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data is available in this paper. Acknowledgments: We are grateful to Alexander V. Martynov (National Museum of Natural History of the National Academy of Sciences of Ukraine) and Janice Peters (Florida A&M University, USA) for their improvements on our draft and comments on the subject. Some data in this research are derived from the database of National Digital-Museum of Animal Specimens. Conflicts of Interest: The authors declare no conflict of interest. Insects 2021, 12, 723 16 of 16

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