Eukiefferiella endobryonia, Imada, 2020

Imada, Yume, 2020, A novel leaf-rolling chironomid, Eukiefferiella endobryonia sp. nov. (Diptera, Chironomidae, Orthocladiinae), highlights the diversity of underwater chironomid tube structures, ZooKeys 906, pp. 73-111 : 73

Eukiefferiella endobryonia sp. nov. Figs 1 , 2

Diagnosis.

Adult male with squama with few (two or three) setae; gonostylus with crista dorsalis; hind tibial comb and tibial spurs reduced, outer spur absent. Pupa lacks precorneal setae and respiratory horns; three anal macrosetae consisting of two thinner inner macrosetae and a normal outer macroseta. Larval body setae short; seta interna with five branches deeply divided to the base; mentum with four pairs of lateral teeth and single, wide, truncate median tooth.

Material examined.

Holotype: USA, VA • 1 adult male (YI-CR-013); Mountain Lake (37.357627 N 80.534448 W); 24-II-2018 (as larva); Y. Imada leg; emerged as adult on 12-III-2018; NMNH.

Paratypes: USA, VA • 2 adult males (YI-CR-009, YI-CR-016) and 3 adult females (YI-CR-010, YI-CR-011, YI-CR-015); Mountain Lake (37.357627N 80.534448W); 24-II-2018 (as larvae); Y. Imada leg; emerged as adults between 12-III-2018 and 28-IV-2018; NMNH.

Other material.

USA, TN • 2 female pupae (YI-CR-001, YI-CR-002), 2 larvae (YI-CR-006, YI-CR-007); Sparks Lane (35.600894N, 83.794004W); 13-XI-2018 (as larvae); Y. Imada leg; NMNH; VA • 1 male pupa (YI-CR-012), 2 female pupa (YI-CR-005, YI-CR-015), 1 pupal exuviae (no voucher), 4 larvae (YI-CR-003, YI-CR-023, YI-CR-024, YI-CR-025); Mountain Lake (37.357627N, 80.534448W); 9-XI-2018; Y. Imada leg, NMNH.

Egg.

Unknown.

First instar larva.

Unknown.

Fourth instar larva.

(N = 4) Body length 3.0 mm. Head capsule dark brown. Body yellowish. Head capsule with frontoclypeal apotome with clypeus without divided by strong suture. Antenna nonretractile, 5-segmented; fourth segment twice as long as third segment; lauterborn organ small; blade as long as flagellum; ring organ in basal third. Premandible with one broad, blunt apical tooth. Mandible with apical tooth longer than first lateral tooth; inner margin smooth, without serrations; seta subdentalis short, peg-like; five very long seta interna with five branches divided nearly to the base, each branch similar in length and width to each other; mola with four long spines. Maxilla without pecten galearis; chaetulae of palpiger lacking; lamellae of galea short; anterior lacinial chaeta apparently short, broad-based, more or less differentiated from other chaetae. Mentum with single median tooth and four pairs of lateral teeth; ventromental plates inconspicuous, without beard beneath. Parapods well developed. Claws of anterior parapods all smooth. Procercus unsclerotized, less than 1.5 times as long as wide, without tooth, spur, or seta; anal setae 5-7. Supraanal seta absent. Anal tubules developed, longer than posterior parapods. Body setae very short and inconspicuous, shorter than one-quarter the length of abdominal segments.

Pupa.

(N = 8) Frontal apotome without frontal seta and warts. Thoracic horn and precorneal seta absent. Dorsocentrals four. Thorax nearly smooth. Wing sheath smooth, without pearl row. T I-II, T VIII, S I and S VIII without shagreen. T II-IX with strong anterior shagreen. S II-VII with weak posterior shagreen. Pedes spurii A and B absent. Caudal spines absent on T II-VIII. S IV-VII female at most with very weak caudal spines. Orally curved hooklets present in uninterrupted rows posterior to caudal spines on T III-V. Apophyses and O setae absent. Segments IV-VIII with very short and weak L-setae. Anal lobe with three unequal anal macrosetae, consisting of two, thinner inner macrosetae and a normal outer macroseta; without median seta, fringe, apical spine.

Adult male.

(N = 3, if not mentioned) Body length 2.9-3.0 mm without antenna. Body color dark brown. Antennal length 0.8 mm. Flagellum plumose, with 13 flagellomeres; apex spatula-shaped, without a strong straight seta; antennal groove in male reaching flagellomere 3; AR 1.1. Eye bare. Temporal setae 2, not clearly separated into inner and outer verticals and postorbitals. Postocular setae present in a single row, only behind eyes. Palpus 5-segmented; palpomere lengths: 55-72, 86-90, 96, 159-159 (N = 1); palpomeres with 3, 4, 5, 0 setae, respectively. Antepronotum well developed with lobes meeting medially at anterior margin of scutum; dorsal anterpronotals absent; four lateral antepronotals; acrostichals absent; six dorsocentrals in a single row. Approximately three prealars. Scutellum smooth with nine scutellars in single row. Supraalar setae present. Wing length 2.3 mm; L/WR 3.01. Wing membrane glabrous, unmarked. Anal lobe small. Costa not extended. Crossvein m-cu absent. Cu1 straight. R4+5 only fused with C at apex. R2+3 present, ending at middle of distance between R1 and R4+5. Cu1 very slightly curved apically at wing margin. Squama with two or three setae. Sensilla campaniformia ca. eight at base of brachiolum, three above setae and eight at apex of brachiolum; 1 on Sc, one basally on R, one near base of R1; and one on FR. Calypter without marginal setae; calyptral fringe absent. First tarsomere of foreleg shorter than fore tibia. Fore coxa not enlarged. Hind tibial comb and tibial spurs reduced; outer spur absent. Pulvilli very faint. Gonostylus hinged to gonocoxite and folded inward. Anal point absent. Anterior margin of transverse sternapodeme convex, phallapodeme and aedeagal lobe normal. Virga absent. Gonocoxite with well-developed inferior volsella. Gonostylus with crista dorsalis; apical spine absent. HR 1.99. Lengths of leg segments and leg ratios as in Table 2 .

Adult female.

(N = 3, if not mentioned) Body length 2.8 mm. Antenna with five flagellomeres; flagellomere lengths (in µm): 56.7, 35.8, 38.2, 45.2, 101.2; with 2, 3, 2, 3, 3 setae, respectively (N = 1). Eye bare. Clypeus with 8 setae. R with two setae, squama with 4-6 setae. Scutellum as in male. Gonocoxapodemes not jointed mesally, well sclerotized. Gonocoxite long, with long and short setae. Tergite IX with two unseparated distinct lobes. Triangular floor under vagina present. Gonapophysis VIII pointed caudally, with two apodeme lobe. Membrane T-shaped. Labia small, bluntly quadrangular, void of microtrichia. Seminal capsule ovoid, darker sclerotized in oral half, without microtrichia. Spermathecal ducts with triangular bulb before separate openings. Cercus normal, length twice as long as width.

Distribution.

North America (US: Tennessee, Virginia).

Etymology.

The species name is a compound word in which three words from Ancient Greek are combined, endo - (ἔνδον), a prefix meaning within, bryon (βρύον), meaning moss, and the suffix - ia (-ία), forming abstract nouns of feminine gender. It alludes to the biology of this species, which live within the case made of mosses.

Remarks.

This species is unique among species of Eukiefferiella in that its pupae lack the precorneal seta. This species can also be distinguished from others in the genus by the following combination of traits: pupa lacks respiratory horns, and has the unique configuration of pupal anal macrosetae (two thinner inner macrosetae, a normal outer macroseta); and larva has a mentum with four pairs of lateral teeth and a single, wide, and truncate median tooth. Any geographic variation in this species’ characters was detectable between the populations sampled in VA and TN.

DNA barcoding.

The results of the species delimitation analyses are summarized in Table 3 . First, BLAST search using the partial COI sequence of voucher YI-CR-001 was executed. This resulted in 98.7 % identical to ' Eukiefferiella sp. voucher BIOUG01648-H02' in GenBank (accession No. KR660601.1) ( Telfer et al. 2015); thus, this sequence was included in the following phylogenetic analyses on the assumption that this specimen may belong to E. endobryonia sp. nov. (Table 1 ). Second, the intra- and inter-specific genetic differentiations were estimated using Bayesian inferences, with the dataset for 28 OTUs. Monophyly of each five species of Eukiefferiella was recovered in the Bayesian phylogeny (Fig. 3 ), as well as E. endobryonia sp. nov. (95 % BPP) together with the above-mentioned sequence data. A Bayesian tree indicated that E. endobryonia sp. nov. was sister to E. dittmari among four species of Eukiefferiella in the dataset with significantly high BPP support (Fig. 3 ). Values of P ID(Strict) for E. endobryonia sp. nov. moderately supported the prediction that this species is correctly identified based on the COI sequence (Table 3 ). Likewise, P(RD) value exceeded 0.05 and hence the clade distinctiveness was supported (Table 3 ). However, Rosenberg’s PAB value was not significant (P = 0.05) and thus the reciprocal monophyly of the clade of E. endobryonia sp. nov. was not supported. Two geographic populations sampled, Great Smoky Mountains (GRSM) and Mountain Lake (ML), formed separate clades and between which genetic divergence among population was substantial (Intra/Inter = 0.12) (Table 3 ), of which values were equivalent to those of the species clade of E. claripennis , composed by the specimens from Europe and Canada (Table 3 ).

Habitat.

Larvae of this species occupied slightly different microhabitats in Mountain Lake, VA (Fig. 4A ) and Sparks Lane, TN (Fig. 4B ). At Mountain Lake, they inhabited a shallow inlet brook flowing into the sink water of the lake. Some leafy and thallose liverworts, including Chiloscyphus ( Jungermanniales : Geocalycaceae ), as well as some pleurocarpous moss species, such as Brachythecium spp. ( Hypnales : Brachytheciaceae ), were abundant there on the upper sides of boulders and cobbles that were exposed to spray and occasionally submerged in water. Fontinalis dalecarlica , a host plant species of E. endobryonia sp. nov., occurred at high densities on the lateral sides of submerged boulders in the stream. As a matter of fact, this seemed to be the only aquatic moss species of which conspicuous amounts were found in this particular stream. I was able to find some white-bodied insect larvae occupying some of the apical shoots of Fontinalis moss plants (Fig. 4C ) simply by looking in the surface layer of slow-moving, shallow water. Interestingly, these larvae apparently resembled the moss capsules enclosed within the bracts of intact plants at first glance (Fig. 4D ). At another locality in TN, the larvae occurred in a stream with fast-flowing water. Some clumps of F. novae-angliae were found growing in this rapidly flowing stream, which were anchored to the sediment of the streambed. The plants bend 50 cm below the water surface in riffle habitats. Similar to observations in the other population, larval and pupal cases occurred at the terminal ends of moss shoots of F. novae-angliae .

Life history.

The life cycle of this new species between late spring and early autumn (May-October) is unknown. This species is likely multivoltine because fourth-instar larvae and pupae were found together at both sites in both April and November. It appears that the larvae were collector-gatherers at the third instar, but became scrapers at the fourth instar (sensu Berg 1995). The third-instar larva restlessly gathered diatoms, which grew on the rims or surfaces of moss leaves. During the later period of the third larval instar, the larva started to dwell on the apical moss shoots and undulated its body among the terminal leaves. Fontinalis leaves are slender, with enrolled margins, and are closely appressed at the tip, forming a semi-enclosed space. At the early stage, a larva showed sinusoidal swimming or undulation behavior ( Brackenbury 2000) within the terminal leaves, where it would later make its case. Approximately five days after colonizing the terminal moss leaves, it developed into the fourth larval instar stage (Fig. 4E ). The fourth-instar larva seemed incapable of living detached from the case due to its limited locomotory habits. When it was removed from its case, the larva attempted to crawl using the anterior half of its body, but was not able to move forward. It spent most of its time feeding on moss leaves. It extended its head and the anterior part of its body outside of the tube to feed, while using its posterior prolegs to maintain contact with a part of its own tube. It grasped the marginal tissues of moss leaves with its mandibles and dragged them back toward its case (Fig. 4F ); simultaneously, silk threads produced from the mouth were extruded with the assistance of the serrated claws of the anterior parapods. The partly grazed leaves were therefore pulled toward the case, which made it easier for the larva to access the surrounding leaf area. The larva repeatedly cut out and fed on the leaves in the bore of the plant in proximity to its case; as a result, ca. 12-20 leaves occurring more or less within ca. 13 mm from the base of the tube were completely consumed (N = 6). The foraging areas were therefore mainly restricted to the region immediately surrounding the tube. This territorial feeding behavior has been reported for many tube-dwelling chironomids (e.g., Darby 1962; Jónasson and Kristiansen 1967; Edgar and Meadows 1969; McLachlan 1977, McAuliffe 1984; Leuchs and Neumann 1990). The larva occasionally defecated, and subsequently immediately ejected the fecal pellet from the end of the tube, which is similar to the behavior of Cladotanytarsus atridorsum (K.) Edw. ( Mackey 1976). The larval fecal pellets were long, ca. half of the body length of the larva, and were loose and cylindrical, which allowed them to easily be released into the water. Under laboratory conditions, younger larvae often used the particles originating from the fecal pellets of mature larvae as tube materials (Fig. 4G ), which means these fecal pellets may also be a source of tube material for younger larvae ( Hirabayashi and Wotton 1999). Judging from the composition of fragments in the fecal pellets, at this stage, the larva largely relied on moss leaves as a food source, which is supplemented with fine amorphous detritus and epiphytic diatoms. Under laboratory conditions, the larva only occasionally withdrew into the tube and engaged in lateral undulations of the body therein.

The larva became less active in the later period of the fourth instar. It scratched the inner surfaces of the leaf margins, not for consuming the leaves, but presumably for strengthening the case wall. As a result of this intensive fabrication behavior, the tissues of the leaves comprising the case became light brown to red in color due to reactions in the plant tissues, whereas undamaged leaves and stems remained green. Approximately half of the larva’s time was spent spinning silk at this point, and the other half was spent staying still. The spinning behavior was stereotyped, regular, and persisted for more than 5 h at a time. The larva lined the interior of the case with silk, which provided a surface with which the claws of the anal prolegs could engage, anchoring the insect within the case. Due to the fabrication and feeding behavior performed in the earlier stages, there were some apertures in the rolled leaf case on the stem-end side. The larva frequently turned around inside the case to strengthen the case’s inside wall. The innermost leaves in the wall, especially at both ends, thus included a thick layer of silk (Fig. 4H ) as a consequence of continuous silk fabrication. Before entering the prepupal stage, debris containing various particles (fecal pellets, diatoms, and strips of moss leaves) was squashed and accumulated at both ends of the case together with a silk mass to seal the end of the tube (Fig. 4I ). At the prepupa stage or later, the case consisted of five or six moss leaves, which were firmly enclosed in silk. The larva molted inside the case and casted off the cuticle; the head capsule and exuvium were thus packed into the posterior end of the pupal case (Fig. 4I ). Pupation occurred with the head oriented toward the distal end of the case, without exception (N = 7) (Fig. 4J ). The pupa rested inside the case throughout most of its development. The pupa then swam toward the surface water and emerged as an adult when its development was completed.

Figures

Figure 1. Eukiefferiella endobryonia sp. nov., adult. A Female head B male antenna C thorax D right wing E hypopygium with tergite IX and with left gonocoxite and gonostylus, in dorsal view with gonostylus (left) and in ventral view without gonostylus (right) F female genitalia, dorsal (left) and ventral view (right) G female tergum IX. Abbreviations (adult). Al: alula; An: anal vein; Ap: antepronotum; Aps: antepronotals; B: brachiolum; C: costa; Ca: coxapodeme; Ce: cercus; Cl: clypeus; Co: cornua; Cp: cibarial pump; Csa: coxosternapodeme; Dc: dorsocentrals; F: fulcrum; Gc: gonocoxite; Gca: gonocozapodeme; Gc IX: gonocoxite IX; Gp IX: gonapophysis IX; Gs: gonostylus; H: humerals; Ivo: inferior volsella; Ll: labial lonchus; No: notum; Pa: prealars; Pha: phallapodeme; Pm: palpal segments; Pn: postnotum; Ps: pseudospurs; Sa: sternapodeme; Sc: subcosta; Sca: seminal capsule; Scts: scutellars; Scu: scutum; Se: spermathecal eminence; Spt: scopula thoracalis; Sq: squama; T IX: tergum IX.

Figure 2. Eukiefferiella endobryonia sp. nov., fourth-instar larva and pupa. Larva (A-C): A general appearance of larva B larval antenna, maxilla and mandible, lateral view C mentum. Pupa (D, E): D pupa, ventral aspect E ditto, dorsal aspect. Abbreviations (larva). Abl: accessory blade; Ap: anterior parapods; As: anal seta; Bl: blade; M: mentum; Mx: maxilla; Pc: procercus; Pm: premandible; Pp: posterior parapods; Ro: ring organ; Sa: supraanal seta; Si: seta interna; Ssd: seta subdentalis; Ta: anal tubules. Abbreviations (pupa). Al: anal lobe; Am: anal macroseta; Ho: orally curved hooklets. Scale bars: 0.1 mm (C), 1 mm (A).

Figure 3. A Bayesian phylogeny based on the COI dataset. Information on the sequences used for this analysis is shown in Table 1. Bayesian Posterior Probabilities were given on each node. Caution is needed to interpret the phylogenetic relationship between and among the members of the species groups (e. g., devonica group, consisting of E. devonica and E. ilkleyensis) included herein, due to the scarcity of available genetic data of the species belonging to the genus Eukiefferiella.

Figure 4. Biology of Eukiefferiella endobryonia sp. nov. A a colony of Fontinalis dalecarlica growing on the sides of pebbles in a gently flowing inlet connected to Mountain Lake, VA, USA (type locality) B a colony of Fontinalis novae-angliae occurring in a rapidly flowing stream at Sparks Lane, TN, USA C early fourth-instar larva, undulating its body in the tube D immature capsule of Fontinalis dalecarlica attached to the stem underwater E fourth-instar larva F fourth-instar larva feeding on a leaf margin of F. dalecarlica G a tube structure of the third-instar larva, which was mainly built from particles from the feces of mature larvae H amorphous, jelly-like silk mass spotted with detritus and diatoms, ripped off of the inner wall of the inner end of the pupal case I a dissected leaf-rolling case, consisting of five leaves and the resident pupa; the leaves used as the case materials are placed in the order of leaf arrangement, with the outermost leaf at the left-most; the innermost leaf (right next to the pupa) contains the head capsule (white arrow) and exuvium of the fourth-instar larva; some debris (pink arrow) and silk mass (black arrow) stuffed in at both ends can be seen J a pupa in its case: the pupal head is oriented toward the distal end of the shoot tip; most of the leaves near the pupal case were consumed by the larva early in the fourth instar stage.