﻿A new North American species of Etainia (Lepidoptera, Nepticulidae), feeding on Arbutus and Arctostaphylos species (Ericaceae)

﻿Abstract Etainiathoraceleuca van Nieukerken, Epstein & Davis, sp. nov. is the second native American species of Etainia Beirne, 1945, and the second known Etainia species feeding on Ericaceae. The species is known from light-collected adults in the USA (California, Arizona) and Canada (Ontario). These were linked via DNA barcodes to larvae that make short leafmines on Arbutus and Arctostaphylos species, then continue feeding in stems and branches, causing damage in nurseries and planted trees in Sonoma and Marin Counties, California. The holotype was accidentally reared from Arbutusarizonica, without observing the damage. Life history and damage are described in detail. Damage in Arctostaphylosuva-ursi found in Washington State probably belongs to E.thoraceleuca, which is a sister species to the European E.albibimaculella (Larsen, 1927).


Introduction
Trees of the genus Arbutus are popular as planted trees for landscaping and gardening in western North America; this applies both to the native Pacific madrone (A.menziesii Pursh) as well as to the European Strawberry tree (A.unedo L.) and the cultivar Arbutus 'Marina', which is of obscure hybrid origin, probably from European stock (San Marcos Growers 2023).Especially the latter is frequently planted as street trees, for example, in San Francisco (Martin et al. 2016).Several Lepidoptera are known as leafminers of Arbutus, especially the heliozelid Coptodisca arbutiella Busck, 1904 and the gracillariid Marmara arbutiella Busck, 1903, which are common on Pacific madrone (Hunt et al. 1992;Eiseman 2022).In 2014 nurseries in Marin County, California reported unknown damage on Arbutus, causing leaf spots and dieback of shoots (Zwart 2017).This damage was first investigated by the late Steven Seybold (University of California -Davis), Drew Zwart, and SVS, who eventually sent larval samples to EvN.DNA barcoding matched these to an undescribed species of Etainia (Nepticulidae) that had been known for some time from adults collected at light, and one specimen that was accidentally reared by D.L. Wagner from material collected on Arbutus arizonica by R.S. Wielgus in 1987.
The Nepticulidae are a medium-large family of very small moths, of which the majority make leafmines as larvae.Globally around 1000 species are known, and 97 named species occur in North America, but many are still unnamed (van Nieukerken et al. 2016; van Nieukerken and Eiseman 2023).
The genus Etainia Beirne, 1945 is one of the smaller nepticulid genera with only 17 named species from Asia, Europe, Africa, and North America (van Nieukerken et al. 2016;Yagi and Hirowatari 2017) and around five undescribed species known in collections.The genus has also been known under the preoccupied name Obrussa Braun, 1915 or as a subgenus of Ectoedemia Busck, 1907.It is one of the few genera of which none of the species make the typical nepticulid leafmines but rather feed in shoots, buds, or fruits.Known hosts belong to Sapindaceae: Acer and Ericaceae, but the hosts for most species are still unknown.The hitherto only known native North American species, E. ochrefasciella (Chambers, 1873), feeds in petioles and buds of sugar maple (Acer saccharum Marshall) and its subspecies (Kulman 1967).The European E. sericopeza (Zeller, 1839), now a widespread alien in North America, feeds in summer in the keys (samaras) of Norway maple, A. platanoides L., and in winter also in a similar fashion as E. ochrefasciella (Jäckh 1951;Emmet and Johnson 1977).In North America it has also been reported to mine petioles of the host in summer (Felt 1930).
The European E. albibimaculella (Larsen, 1927) feeds on bearberry (or kinnikinnick), Arctostaphylos uva-ursi (L.) Spreng., and makes mines that start in the leaf but then immediately enter the shoots (Adamczewski 1947).The specimen of the new species, taken in Almonte, Ontario, in an area where bearberry is common, was first misidentified as E. albibimaculella, which as a result was wrongly recorded as Holarctic and Canadian (van Nieukerken et al. 2016;van Nieukerken 2018).
We here describe the new species and compare it with congeners and other similar North American species.The other two North American species were previously treated by Wilkinson and Scoble (1979).

Material
We list here material without coordinates, more detailed specimen data are provided in GBIF dataset https://doi.org/10.15468/espa8k.Abbreviations for depositories, etc.(often combined with catalog numbers)

BIN
Barcode Index Number (Ratnasingham and Hebert 2013) Larvae were collected by cutting off ends of symptomatic branches of various hostplants and dissecting them using a scalpel under a Leica MZ75 stereomicroscope.Larvae were teased from their tunnels with a pin, and frozen before shipment to CDFA labs, or alternatively kept in ethanol 80%.
Pupae were recovered from the duff within the adult enclosures (above) and later from beneath heavily infested plants in the landscape.Cocoons were found about 5 cm beneath the surface of the duff, usually sandwiched between fragments of two dead leaves to which they were lightly attached.
We further added data obtained from observation platforms iNaturalist, Bug-Guide, and Barcode of Life Data Systems.

Morphology
Genitalia were prepared according to standard procedures-those by DRD using Canada balsam as embedding medium, those by EvN usually including DNA extraction-and using Euparal as embedding medium; see earlier papers (van Nieukerken 1985;van Nieukerken et al. 2010).Larval slides were prepared in the same way.

Measurements
Measurements of genitalia were obtained from digital images, using calibrated scaling in the Zeiss AxioVision software; we used a 20× objective for male genitalia and 10× or 20× for female genitalia.Capsule length was measured from vinculum to middle of pseuduncus; valva length from tip of posterior process to ventral edge, excluding the sublateral process; phallus length was measured along the sclerotized tube, from tip, excluding carinae.Total corpus bursa length was measured from where the ductus bursae widens into the corpus bursae to anterior edge of bursa.Genitalia measurements are rounded off to the nearest 5 μm.Forewing length was measured from tip of fringe to attachment on thorax, with a Zeiss SV11 stereomicroscope at a magnification of 20×.Antennal segment counts include scape and pedicel; they were counted on photographs or directly under the same stereo microscope.Larval measurements of potential 2 nd , 3 rd , and 4 th instars, mounted on slides, were from San Rafael, California (Figs 22,23,26) (S.Seybold, see below) and those measured from ethanol by MEE were 1 st to 4 th instars (n = 21) and from San Rafael and Sonoma, California 24,25,27,28; see below).

Photographs
Photographs of moths were made with an AxioCam MRc 5 digital camera attached to a motorized Zeiss SteREO Discovery V12, using the Module Extended Focus and Zeiss AxioVision software to prepare a picture in full focus from a Z-stack of ca 10-40 individual photos.Genitalia were photographed with an MRc 5 camera on a manually operated Zeiss Axioskop H, without using extended focus.Photographs were edited with Adobe Photoshop (various versions), avoiding changes to the real object, but backgrounds were cleaned of excess debris and artifacts by using the healing brush and clone tools; tone and contrast are adjusted, and some sharpening was used.Larvae in fluid, as well as dry cocoons, pupal skin, and a parasitoid, were photographed with a Leica MZ 16 using LAS IV Z-stack, as above, and scanning electron microscopy of the larvae was done using a Vega 3 Tescan with normal vacuum; the samples were sputter-coated with gold palladium.

DNA barcoding
Our methodology has been described in other papers ( van Nieukerken et al. 2012;Doorenweerd et al. 2015Doorenweerd et al. , 2016)).We present a neighbor-joining tree, with KP2 distances, of the selected taxa, made with tools provided by BOLD Systems (Ratnasingham and Hebert 2007).The DNA barcode data as used here are given in detail in the public BOLD dataset DS-ETAARB (Etainia Arbutus) (https://doi.org/10.5883/DS-ETAARB),including GenBank accession numbers.

Hostplants
Hostplant names follow Catalogue of Life (Hassler 2023) and the Flora of North America (Parker et al. 2009;Sørensen 2009).The larvae were recovered from various manzanita species (Arctostaphylos).Given the large number of species in the genus, particularly in California, we did not attempt to identify the species of hostplant, but some of the cited observations do include species identifications.See Parker et al. (2009) for more information.Where species names are provided, we cannot guarantee that all species identifications are fully correct..31006, 31007, 31012-15 • 20  Diagnosis.Etainia thoraceleuca is easily recognized by the combination of a white thorax and the silver markings: a fascia and costal plus dorsal spot.Some Stigmella species have a similar pattern, but can be recognized by the distinct collar, comprising lamellar scales.Most similar are some species of Acalyptris, including the eastern A. thoracealbella (Chambers, 1873).This species has the pattern not so silvery, the antennae are paler, and the wings are narrower; moreover, the distribution does not seem to overlap much, but genitalia should be checked when in doubt.The male genitalia are characteristic of the genus Etainia by the valval apodemes, absent uncus, and structure of the phallus; it differs from E. ochrefasciella and E. sericopeza by the different shapes of the valva and gnathos, and the latter are very wide in E. sericopeza and very narrow in E. ochrefasciella.The female genitalia differ especially by the different structure of tergite 8.
Female genitalia .Abdominal end broadly rounded, anal papillae a narrow band with more than 50 setae in total; T8 with pointed posterior margin; on T8 three transverse broken rows with groups of socketed setae, posteriorly two connected groups of ca 21-25 setae each; medially two widely separate groups of 12-13 setae at either side; anteriorly two groups of ca 14-17 setae.Anterior apophyses widely separate, with curved tips; posterior apophyses straight.T7 with medial indentation in posterior margin.Corpus bursae total length ca 710-855 μm.Ductus bursae with a paired sclerotized structure near cloaca and group of small spines laterally, more anteriorly; corpus bursae with paired elongate reticulate signa, usually different in length, longest 520-550 μm; ca 10-12 cells wide, shortest 360-425 μm, ca 10-17 cells wide.Ductus spermathecae with 2 indistinct convolutions.
Egg.In the few examples seen on leaf underside, the usual domed egg scale of Nepticulidae.When the mine develops, the egg is more or less in the center of the leaf spot.

Life cycle.
Eggs are apparently laid singly on the underside of leaves in late summer to early fall, where they remain quiescent for several weeks.In late fall, the larvae hatch and begin burrowing into leaf tissue.On the oviposition site there develops a red to black stained leaf spot; from there the larvae make a thin, linear mine running along the lateral and midveins of the hostplants' leaves, often very straight or sometimes with a few loops; the frass line is central and almost completely fills the gallery; the mined area soon turns red to black.Larvae continue burrowing through the petiole and into subtending twigs where they mine phloem tissues.After feeding in the twig cambium for several months, larvae bore their way out of the twigs and drop into the leaf litter beneath the plant.The exit holes are little slits in the twig epidermis, resembling those of other  44).Initial damage symptoms appear as inconspicuous black spots on the hostplant leaf blade, caused by a larva tunneling in a wandering circular pattern (Fig. 41).The circles gradually enlarge until the larva encounters a lateral leaf vein.The larva then normally follows the lateral vein down to the midvein, through the petiole .Damage by the larvae can be seen on the undersides (and sometimes the top) of the leaf as a thin but conspicuous red to black line marking the tunnel.Sometimes a larva will follow the lateral vein to the edge of the leaf, and then circle toward the leaf center until it again finds a lateral vein.Once the larva reaches the thicker tissues of the petiole and begins boring into the twig's cambial tissues the line gradually disappears.As larvae from many leaves migrate into subtending twigs to feed, their numbers and associated damage become concentrated, damaging the cambium to such an extent that the distal portions of the twig are frequently killed.These branches wilt and die rapidly, leaving the wilted end of the twig to droop, shrivel, and then harden into a shepherd's crook.Even when twigs are not killed, the bark splits and callus tissue will form on the twigs, disfiguring and sometimes distorting it.In severe cases, young Arbutus 'Marina' trees have been killed, and older trees rather severely disfigured, with less than 50% live canopy remaining.Damage to native manzanita and madrone does not appear to be as severe.
Parasitoids.As might be expected for a moth in its native range, parasitoid wasp pupae have been found inside the mines on occasion.One Chalcidoidea parasitoid (most likely Eulophidae, Entedoninae, C. Eiseman pers.comm.)emerged in summer 2023 from cocoons from Sonoma (near Napa Co.) found by SVS on 11 May 2023; it made a circular exit hole on a broad cocoon surface (Fig. 53).A moth emerged from another cocoon from the same date and location during the same summer.DNA barcodes (Fig. 60).We have DNA barcodes from eleven specimens, four larvae and seven adults: seven full barcodes belonging to BIN BOLD:ACK1467 with an average KP2 distance of 0.39%, and a maximum distance of 0.83%, and three barcodes of the Canadian specimens belonging to BIN BOLD:AEO1837 (no variation) at a distance of 1.5% to the nearest Californian sequence (Fig. 58).Unfortunately, we failed to amplify COI from the holotype.
The identical barcodes of adults and larvae undoubtedly show that the described larvae and damage belong to the same species as the adults.
Remarks.The specimens from Canada differ in having a completely orange head and in their female genitalia; in both specimens the signa are almost equal length, 360 μm.In a Californian female specimen one of the three rows of setae on T8 seems to be missing.The genitalia of the male Canadian specimen could  59 not yet be examined.For now, we consider the Canadian specimens as belonging to the same species, but further study and material is required to evaluate the possibility of hidden diversity.
Etymology.The specific name, thoraceleuca, a noun in apposition, is derived from the Greek noun thorax (breastplate) and adjective leukos (white), referring to the characteristic white thorax.

Phylogeny
In a maximum-likelihood analysis of Nepticulidae based on a maximum of eight genes (to be published elsewhere), E. thoraceleuca groups with E. albibimaculella and the eastern Palearctic E. capesella (Puplesis, 1985) as sistergroup to all other Holarctic representatives of Etainia, which are probably all associated with Acer (Sapindaceae).The grouping of E. albibimaculella and E. capesella was also observed earlier (Doorenweerd et al. 2016), and since both E. thoraceleuca and E. albibimaculella feed on Ericaceae, it may indicate that E. capesella is also an Ericaceae feeder.
The sister-group relationship between E. thoraceleuca and E. albibimaculella may suggest a scenario where the common ancestor invaded North America from Asia, where it continued on the same hostplant, Arctostaphylos uva-ursi and from there invaded the mountains in the West and expanded its host range to include the abundant other Ericaceae hostplants already present.

Distribution
The three Canadian records, far away from most records from the western USA, suggest that the species may have a continuous distribution with the northern Arctostaphylos uva-ursi, which has a very wide distribution throughout Canada and the northern and western United States.It is remarkable, however, that this relatively large and conspicuous nepticulid has not been recorded anywhere else in Canada, and also not in the extensive Canadian Malaise trapping programs (Hebert et al. 2016).In fact, E. thoraceleuca has only been collected twice in a Malaise trap, the specimens cited here as BIOUG08839-B12 from San Diego County and BIOUG92695-A02 from Santa Clara County.The apparent rarity is comparable to that of the European E. albibimaculella, for which records outside Finland and Sweden are extremely rare (van Nieukerken and Johansson 1990;Roweck and Savenkov 2002;Mazurkiewicz 2007;Kopp 2010;Nel et al. 2020), despite the likewise large distribution of bearberry in Europe and Asia.

Hostplant relationships
The genera Arbutus and Arctostaphylos are closely related and grouped in the subfamily Arbutoideae (Kron et al. 2002), and many insect species share these genera as hosts.This is also the case for several leafminers: Coptodisca arbutiella, Marmara arbutiella, Coleophora glaucella Walsingham, 1882, Epinotia nigralbana (Walsingham, 1879), Epinotia terracoctana (Walsingham, 1879), and an undescribed Stigmella species (Eiseman 2022).The oligophagy of Etainia thoraceleuca thus follows this common pattern.It should be searched for on Arbutus xalapensis in New Mexico and Texas and is likely to be found on many more Arctostaphylos species.

Impact
As Etainia thoraceleuca is indigenous in California, its occurrence on cultivated plants was to be expected and by itself not of much concern.In some cases, however, the damage made by the insect can become a nuisance, but the use of the neonicotinoid imidacloprid in many nurseries (personal communications to SVS) has probably prevented this from becoming problematic.This, however, is in no way a plea to use these pesticides against this insect, as the damage caused by neonicotinoids to insects, especially bees, is too well known (Pisa et al. 2021).It is better to allow the native parasitoids and predators to do their work.Care should, however, be taken to avoid distributing infested plant material outside the native area, to avoid the possibility that E. thoraceleuca could become established elsewhere and might become invasive.
Distribution (Figs 58, 59).Most records are from California, where E. thoraceleuca has been found over much of the coastal ranges of California and collected from Del Norte County in the extreme northwestern part of the state to as far south as San Diego County, in adjacent Oregon (leafmines, Josephine Co., Rough and Ready Botanical Wayside, 42.0959, −123.6831;C. Eiseman pers.comm.), in Arizona in the Huachuca mountains (type locality), Chiricahua

Figure 60 .
Figure 60.Neighbor-joining tree of DNA barcodes (mt gene COI-5P) of Etainia species, using the Kimura distance model.Acalyptris scirpi (Braun, 1925) is used as outgroup.Labels show species name, Sample ID, life stage, Country, State or Province and BIN (Barcode Identification Number).
CASENT California Academy of Sciences, Entomology, San Francisco, CA, USA; ; BIOUG Biodiversity Institute of Ontario, University of Guelph, ON, Canada; BOLD Barcode of Life Data Systems (http://www.barcodinglife.com/);Methods Adults were usually collected at light by various collectors.The holotype was reared accidentally.SVS collected adults by setting five-gallon Arbutus 'Marina' (bush) trap plants out nearby infected trees from 2900 Wild Turkey Run, in the Bennett Valley area south of Santa Rosa, California.Once trap plants showed symptoms of infestation, they were transported back to a holding area, where the plants had plastic funnels constructed around their bases (0.013 mm clear polycarbonate sheeting cut and glued into a 68 cm tall funnel, with a top opening of 46 cm and a bottom opening of 25 cm).Nets measuring 175 cm × 71 cm were constructed using a Singer model 4423 sewing machine, of white organdy (JoAnn Fabrics and Crafts, Rohnert Park, CA), white polyester thread, and cinched with black 4 mm diameter parachute cord.These nets were placed over the entire plant and funnel assembly in April of 2020 and checked twice weekly.