Three new species of Fancy Case caterpillars from threatened forests of Hawaii (Lepidoptera, Cosmopterigidae, Hyposmocoma)

Abstract The endemic Hawaiian moth genus Hyposmocoma includes 348 described species and perhaps twice as many that remain undescribed. The genus is unusual within Lepidoptera in that its larvae create distinctive silk cases in which they perambulate while protected and camouflaged. An extraordinary diversity of case types exists, and to date more than ten different types have been identified, each corresponding roughly to a separate evolutionary lineage. In this study, we describe three new species of Hyposmocoma: Hyposmocoma ipohapuu sp. n. from Big Island, Hyposmocoma makawao sp. n. from Makawao Forest Reserve in Mauiand Hyposmocoma tantala sp. n. from Mt. Tantalus, Oahu, all of which produce tubular purse cases during their larval stage. We also describe the female of Hyposmocoma inversella Walsingham, which was previously undescribed, and re-describe two closely related species, Hyposmocoma auropurpurea Walsingham and Hyposmocoma nebulifera Walsingham, neither which have been formally described in recent years. We present for the first time, primer sequences for a 705 bp fragment of CAD, designed for Hyposmocoma and relatives. The molecular phylogeny based on mitochondrial and nuclear loci demonstrates that all are distinct species. The discovery of a new, endemic species from Mt. Tantalus, an area with many invasive species, suggests that even relatively degraded areas in Hawaii would be worthy of active conservation efforts.

. This is part of an ongoing project to document the extraordinary diversity and ecology of Hyposmocoma across the Hawaiian Islands (e.g. Rubinoff 2008;Rubinoff and Haines 2005;Rubinoff and Schmitz 2010;Schmitz and Rubinoff 2008;).

Specimen preparation, dissection, and description
Genitalia preparation techniques and morphological terminology follow previous work on Hyposmocoma (e.g. Rubinoff and Haines 2006;Schmitz and Rubinoff 2008;. The abdomen of the dried moth was placed in a 20% KOH solution at room temperature overnight. When fats were not fully dissolved, the KOH solution and abdomen was heated on a hot plate. Genitalia were dissected and transferred to a solution of lactic acid with Orange G to stain hard cuticle. Membranes of the female genitalia were subsequently stained with Chlorazol Black, fixed in a sequence of 30%, 70%, and 95% ethyl alcohol, and mounted on slides with Euparal. Genitalia of two specimens, a female of H. ipohapuu and a male of H. makawao were prepared at the beginning of this study and were stained only with Orange G. Forewing and case lengths were measured with a scale in the ocular of a Leica MZ16 stereomicroscope. Adult moths were photographed with a Canon D50 digital camera mounted to the microscope; genitalia were photographed with an Olympus QColor3 Camera mounted on an Olympus SZX10 stereomicroscope. Types of previously described Hyposmocoma were examined from the collections of the Bernice P. Bishop Museum (BPBM) and the University of Hawaii Insect Museum (UHIM) on the University of Hawaii Manoa campus. Non-type specimens were examined from these museums and the US National Museum of Natural History (USNM). Types of the new species described here are deposited in the UHIM. All specimens in this study were reared from larvae, which were fed carrots and fish flakes and stored in Petri dishes until emergence.
Genomic DNA was extracted from all specimens using the DNeasy TM animal blood and tissue extraction kit following recommended protocols (Qiagen, Inc., Valencia, CA). The tissue was digested at 56° C for 24 hours, 200 ml of EB buffer was used to elute the DNA and extracts were stored at -20° C. COI was amplified in a single fragment: the forward primer Jerry (CAA CAT TTA TTT TGA TTT TTT GG) and reverse primer Pat-2  (TCC AAT GCA CTA ATC TGC CAT ATT A ;Simon et al. 1994), and for EF-1α, a single fragment with the forward primer Coma (GGC CCA GGA AAT GGG CAA AGG) and reverse primer Toes (GGA GTC WCC AGC KAC GTA ACC; Rubinoff 2008), and for CAD the primers Cue (GGG ATT TAG CAA AAT TCA AAC GC) and Eh (CCT ATC GCT CAT ATC ATA ATC GG). The primers Cue and Eh were not published previously and thus described here. Amplifications were performed in a total volume of 25 μl. Thermal profiles for COI and CAD (94°C for 1 min; 51°C for 1 min; 72°C for 2 min), differed from EF-1α (94°C for 1 min; 53°C for 1 min; 72°C for 2 min). Each reaction started with 2 min of denaturation at 94°C, for 34 cycles, and followed by a final elongation for 12 min at 72°C. All PCR-products were visualized on 1% agarose gel and purified using QIAquick® spin columns (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. Sequencing was conducted with an Applied Biosystems 3130xl Genetic Analyzer at the Greenwood Molecular Biology Facility, Pacific Biosciences Research Center, the University of Hawaii at Manoa. For each sample, both sense and anti-sense strands of PCR products were sequenced. Multiple sequence alignments were conducted using MAFFT ver. 6.843 (Katoh 2010) and assembled in Geneious ver. 5.1 (Drummond et al. 2010). Genbank accession numbers are listed in Table 1. Phylogenetic analyses were conducted with maximum likelihood (ML) as implemented in GARLI 1.0 (Zwickl 2006), and in a Bayesian framework with the software MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck 2003). We used jModelTest (Posada 2008) to determine the best substitution model which was always the General-Time-Reversible (GTR) model (Lanave et al. 1984;Tavaré 1986), incorporating among-site rate heterogeneity modeled according to a gamma (G) distribution (Yang 1994) while allowing for a proportion of invariable sites (I) (Gu et al. 1995). One thousand ML and bootstrap tree searches were conducted as implemented in GARLI. To expedite tree searches, we used Grid computing (Cummings and Huskamp 2005) through The Lattice Project (Bazinet and Cummings 2009). GARLI analyses were conducted using the Lattice Grid portal (Bazinet and Cummings 2011). Bayesian analyses were conducted for 5 × 10 7 generations with default settings and 3 × 10 7 generations were treated as burn-in and removed. Chain convergence was assessed with MrBayes and AWTY Online (Wilgenbusch et al. 2004).

species descriptions
Hyposmocoma auropurpurea Walsingham, 1907 http://species-id.net/wiki/Hyposmocoma_auropurpurea Figs 1,11,18 Diagnosis. This species is unique among species of Hyposmocoma because it has metallic purple wings with a narrow, diagonal orange band near the wing apex.

Re-description.
Male. (n = 2; Fig 1). Forewing length 4.8-5.0 mm. Head with a mixture of copper and metallic purple colored scales arranged radially from compound eye. Haustellum with light brown scales. Maxillary palpus reduced. Labial palpus curved with copper and metallic purple colored scales, scale color similar on all surfaces of palpus. Antenna brown with a mosaic of metallic purple scales. Thorax mostly copper; dark brown/ purple scales present along anterior margin. Foreleg coxa with brown and metallic purple scales; femur, tibia, and tarsomeres mostly dark brown. Midleg as foreleg, but spurs covered in a mixture of dark and light-brown scales. Hindleg as midleg, but longer with long hairs on dorsal margin of tibia. Forewing metallic with a narrow diagonal orange band near wing apex. Diffuse orange patch in proximal region of FW along anal margin. Fringe orange and brown/metallic purple, longer scales tending to be brown/metallic purple, shorter ones orange. Hindwing brown with brown fringe. Abdomen dorsally dark brown; ventrally metallic brown/purple, with tuft of long dark brown scales covering lateral surface of genitalia.
Larval case (n = 7). Dark brown, smooth, 7-9 mm in length and 1.7-2 mm wide. Biology. Case-making larvae were collected from October to April. Larvae were typically found in leaf litter, but in some cases were found on leaves such as Pittosporum sp. (Pittosporaceae).

Hyposmocoma inversella
scales. Maxillary palpus reduced. Labial palpus curved with copper colored scales, scale color similar on all surfaces of palpus. Antenna brown with a mosaic of copper scales. Thorax mostly copper; dark brown scales present along anterior margin. Foreleg coxa with brown and copper scales; femur, tibia, and tarsomeres mostly dark brown with light brown ring at distal margin of femur, tibia, and tarsomeres I-V. Midleg as foreleg, but spurs covered in a mixture of dark and light-brown scales. Hindleg as midleg, but longer with long hairs on dorsal margin of tibia. Forewing brown, two transverse orange fascia form a "V". Proximal orange band widens along anal margin, distal band uniform in width. Fringe orange and brown, longer scales tending to be brown, shorter ones orange. Hindwing brown with brown fringe. Abdomen dorsally dark brown; ventrally copper, with tuft of long dark brown scales covering lateral surface of genitalia.
Male genitalia. Right brachium of uncus sickle shaped, slender, heavily sclerotized, widening at 1/2 length, slightly twisted to left. Left brachium small, not sclerotized. Tegumen wide and sclerotized. Four long thin setae along dorsal margin of left valva, three shorter setae at same location of right valva. Left valva slightly wider than right. Dense row of fine, hair-like setae disposed along inner ventral margin of valva. Phallus stout, blunt tipped, heavily sclerotized, open ventrally, and bent ventrad at about 1/2 of length; vesica without spines or cornuti. Anellus with two symmetrical rounded lobes with short setae.
Larval case. (n = 2). Dark brown, smooth, 7.8-8.5 mm in length and 1. Diagnosis. Hyposmocoma ipohapuu has a forewing pattern that differs from any other described species in the genus. A wide pale yellow band is present along the anal margin of the forewing and curves proximally at the wing margin towards the costa. A small, round, dark brown mark is present approximately two thirds of the way from the wing base to the apex. Description. Male (n = 2; Fig. 3). Forewing length 5.0 mm. Head with pale brown scales radiating from compound eye. Haustellum with a mixture of light and dark brown scales. Maxillary palpus reduced. Labial palpus recurved and covered in pale brown scales, which are dark laterally. Antennal flagellum pale brown, with a ring of dark brown scales extending from the proximal margin of each flagellomere. Thorax mainly pale brown, dark brown at cervical margin and near posterior margin of notum. Foreleg dark brown with pale brown ring at distal margin of femur. Midleg as foreleg, but with pale brown ring at distal margin of tibia and tarsomeres I-V, spurs pale brown. Hindleg as midleg but longer with long pale brown hairs. Forewing pale brown, with dark brown band extending from costal margin of wing base to apex, mark forming a convex arch near apex. A separate brown band extends along anal margin of forewing. Fringe pale brown. Hindwing and hindwing fringe grayish brown. Abdomen dorsally dark brown; ventrally pale brown with a tuft of long pale brown scales on either side of genitalia.
Female. (n = 1). Externally as male, but with longer forewing length (5.8 mm) and small dark brown mark medially below dark band on forewing.
Larval case. (n = 5; Fig 7). The mature case is 5.9-6.6 mm in length and 1.  (5): same data as holotype. All pinned specimens and slides deposited in the UHIM.
Specimens sequenced. Three specimens from Big Island, Hawaii, USA (UH log number DR08K8C), extraction codes DN121, DN1321 and KY2. This species was tentatively called "Hsp69" before given this formal name.
Biology. Case-making larvae were collected on the abaxial surface of old fronds of a Hawaiian tree fern (Hāpu'u, Cibotium glaucum (Sm.) Hook. & Arn., Cibotiaceae) in the rain forest at Volcano Village near Hawaii Volcano National Park, Hawaii (Big) Island. The habit of residing in old tree fern fronds, still attached to the stump, is typical of several purse case species including H. filicivora Meyrick, 1935. Distribution. Known only from Volcano Village, Hawaii Island. Probably restricted to the rainforest areas around Hawaii Volcanoes National Park, where it appears to be uncommon. The elevation for the type locality is approximately 1200 m.  1; Fig 4). Forewing length 4.8 mm. Head brown with iron-red scales near outer margin of eye; scales large near vertex. Haustellum pale brown. Maxillary palpus reduced. Labial palpus curved with pale brown scales, scales dark brown along lateral margin of labial palpus. Antennal flagellum dark brown along dorsal surface, lighter brown ventrally. Thorax reddish brown, laterally brown with patches of lighter brown scales below forewing. Foreleg and midleg with brown scales, scales dark brown laterally. Hindleg same as midleg, but with long scales along dorsal margin. Spines on legs light brown. Forewing dark brown with a single, wide fiery redbrown transverse fascia one fourth from the base of the wing to apex and narrowing towards costal margin. Abdomen covered in dark brown scales dorsally, light brown scales covering ventral surface.
Female. (n = 1). Same as male, but with forewing length 4.9 mm. Larval case. (n = 7; Fig 8). The case is 4.1-5.0 mm in length and 1.2-1.6 mm wide, smooth with banding that follows the length of the case. Two wide, dark bands form a "V" that crosses over the central region of the case. Specimen in perfect condition. Paratype: 1♀: same data as holotype, but emergence date 13-Feb-2009, abdomen missing. Cases (7): same data as holotype. All specimens stored in the UHIM.
Specimens sequenced. Two specimens from Maui with UH log number DR-08K12A, extraction codes DN114, DN1320. The specimen from which extract DN114 was taken was tentatively called "Hsp67" before being given this formal name.
Etymology. This species is named "makawao" after its type locality, Makawao Forest Reserve, Maui.
Biology. Case-making larvae were collected at Makawao Forest Reserve (MFR), Maui. Samples were collected using a beat-sheet, placed under branches of the koa tree (Acacia koa Gray).
Distribution. Known only from the MFR, which harbors an extraordinary diverse natural fauna and flora, and is the only known locality for several species of Hyposmocoma, including H. domicolens (Butler, 1881), H. molluscivora Rubinoff & Haines, 2005, H. opuulaau Schmitz & Rubinoff, 2011, H. pukoa Schmitz & Rubinoff, 2011, and H. pupumoehewa Schmitz & Rubinoff, 2011. Unfortunately, the native habitat has been in sharp decline, even during the short course of this project, due to ongoing damage from invasive ungulates. If conservation action is not soon taken, it is likely that many of the rare plants and animals that remain in this mixed mesic forest will disappear.

Hyposmocoma nebulifera Walsingham, 1907
http://species-id.net/wiki/Hyposmocoma_nebulifera Figs 5,9,12 Diagnosis. Hyposmocoma nebulifera is similar to H. rubescens from Kauai, but differs in having a larger dark brown C-shaped forewing mark, and much smaller spurlike specialized setae on the valva. It is also similar to H. ekemamao but is larger, and has two central round spots on the forewing, while H. ekemamao only has one.
Re-description. Male. (n = 3; Fig 5). Forewing length 5.8 -6.0 mm. Head light brown with scales near outer margin of eye, scales large near vertex. Haustellum pale brown. Maxillary palpus reduced. Labial palpus curved with pale brown scales, scales dark brown at terminus. Antennal flagellum light with dark brown bands. Thorax light brown, with lighter brown scales on tegula. Foreleg and midleg with brown scales and bands of light brown. Hindleg same as midleg, but with long scales along dorsal margin formed into a brush-like patch. Spines on legs light brown. Forewing light brown with a C-shaped dark brown mark 1/3 of distance to forewing apex, dark brown spot at base of forewing. One dark brown mark with light border at center of wing, another mark about 1/3 distance between first mark and apex. Abdomen covered in brown scales.
Distribution. Known only from the Waianae Mountain Range, Oahu. With nearly thirty described species of Hyposmocoma (Zimmerman 1978), the Palikea preserve in the Waianae mountains has a very high diversity of Hyposmocoma, and the area is also one of the best-preserved on Oahu. The rich species diversity of Hyposmocoma may reflect the diversity throughout the island before human colonization. Diagnosis. Hyposmocoma tantala is similar to H. nebulifera, but differs in having a dark forewing background color and much thicker dark forewing markings. The male genitalia has large sclerotized spines on the left valva that are absent in H. nebulifera.

Hyposmocoma tantala
Description. Male. (n = 1). Forewing length 5.5 mm. Head light brown with light brown scales near outer margin of eye; scales large near vertex. Haustellum pale brown. Maxillary palpus reduced. Labial palpus curved with pale brown scales, scales dark brown at terminus. Antennal flagellum light with dark brown bands. Thorax light brown, with lighter brown scales on tegula. Foreleg and midleg with brown scales and bands of light brown. Hindleg same as midleg, but with long scales along dorsal margin formed into a brush-like patch. Spines on legs light brown. Forewing light brown with a large dark brown mark extending from costal margin 1/3 of distance to forewing apex and a smaller dark brown mark at 2/3 of distance to apex. Abdomen covered in brown scales.
Male genitalia (Fig. 13). Right brachium of uncus thick and curved ventrad, smooth, gradually tapering, slightly twisted to left. Left brachium small, not sclerotized. Tegumen wide and sclerotized. Shape of valva largely symmetrical, but left valva with three long tapered narrow spurlike setae along dorsal margin near apex; right valva without large sclerotized setae. Dense row of fine, hair-like setae disposed along inner ventral margin of both valvae. Phallus stout, blunt tipped, heavily sclerotized, open ventrally, and bent ventrad at about 1/2 of length; vesica without spines or cornuti. Anellus with two symmetrical rounded lobes with fine setae.
Female. (n = 2; Fig 6). Externally as male, but with larger wing span (6.2 mm). Female genitalia. (Fig 16). Papillae anales short and setose. Anterior and posterior apophyses thin and straight, posterior apophyses slightly longer than anterior apophyses. Ostium bursae heavily sclerotized, externally protruding, and C-shaped curled left. Ductus bursae long and of small girth. Corpus bursae roughly kidney-shaped, with light scobination; signum absent. Apical margin of tergum VIII with median emargination.   Fig. 19  Etymology. This species is named "tantala" after Mt. Tantalus, from where the type specimen was collected. While Mt. Tantalus has experienced a tremendous amount of destruction from invasive species in the past century, it has historically been a locality with very high endemism, and the type locality of several other endemic insects, including flies (Hardy 1960;1964), beetles (e.g. Liebherr and Zimmerman 2000), and at least two other species of Hyposmocoma (Zimmerman 1978). We wish to recognize this historically noteworthy site and draw attention to the fact that some endemic species, worthy of conservation, still persist even after long periods of disturbance.
Biology. Adults were reared from case-making larvae collected on bark of a damp dead tree covered partially with lichen.
Distribution. Known only from Mt. Tantalus, Oahu.

Conclusion
Our morphological investigation coupled with molecular sequence data supports the separation of all three species described as new in this study. Interspecific genetic divergence in COI for sister-species for other Lepidoptera range from slightly less than 1% to nearly 4% (e.g. Landry et al. 1999;Rubinoff and Powell 2004;Sperling and Hickey 1994, but see also Mitter et al. 2011), suggesting that species of Hyposmocoma studied here exhibit relatively high levels of interspecific divergence. This may be due to lineage-specific rates of evolution, and/or differential rates of colonization and extinction in the Hawaiian islands. Future research will focus on the evolutionary processes behind the distribution and diversity of purse-cased Hyposmocoma and their relatives. While we have sampled Hyposmocoma broadly on all Hawaiian islands, all species in this study and those previous appear to be largely restricted to one volcano on one island. Unfortunately, restricted ranges pose challenges for conservation since many species are vulnerable to extinction because of widespread habitat destruction. Mount Tantalus on Oahu, the only known locality for H. tantala, is the type locality for endemic flies, beetles and at least two other species of Hyposmocoma. The area is heavily infested with invasive species, but does retain some endemism, though the native habitat is still declining. The discovery of a new, endemic species from Mt. Tantalus suggests that even relatively degraded areas would be worthy of active conservation efforts to control invasive species. The same is true for the Makawao Forest Reserve on Maui that straddles an area between rainforest and dry forest, and thus supports a particularly diverse, highly unique endemic flora and fauna. This forest reserve is also the type locality for six species of Hyposmocoma. As such, native species in these refugia continue to decline, and this must include endemic species of Hyposmocoma, all of which are found nowhere else. We hope this information will encourage more active conservation, not only to protect these moths, but also the immense diversity of geographically restricted species displaying amazingly specialized life histories and the remnant native habitats that sustain them.