Raphia Hübner is a small genus of the Holarctic region, with a single African species (Poole 1989) questionably congeneric. As the sole genus currently comprising the Raphiinae, the phylogenetic placement of Raphia has an interesting history. Most early works included the genus in older concepts of the Pantheinae. Smith (in Smith and Dyar 1898) excluded Raphia from the Pantheinae, but remained uncertain of its affinities within the Noctuidae. Hampson similarly excluded Raphia from the Pantheinae (Hampson 1913) and Acronictinae (Hampson 1909), and although never published, Raphia would presumably have been included in Hamspon’s volume covering “Ophiderinae” (Kitching 1984), a catch-all subfamily for noctuoids with fully quadrifine hindwing venation and lacking other specialized features emphasized by Hampson. Forbes (1954) and Franclemont and Todd (1983) maintained Raphia in Pantheinae, and it was not until recent times that Beck (1996) proposed a separate subfamily to accommodate Raphia. Two additional genera were recently thought to be related to Raphia: Diloba Boisduval (Fibiger et al. 2009) and Aon Neumoegen (Fibiger and Lafontaine 2005), the former resulting in a family-level synonymy of Raphiinae under Dilobinae (Fibiger et al. 2009). Molecular study and re-assessment of morphological traits has since shown that Raphiinae and Dilobinae are best retained as valid subfamilies (Zahiri et al. 2013). Similarly, Aon has subsequently been reclassified as belonging to Erebidae: Hypocalinae (Lafontaine and Schmidt 2010).

Three Raphia species occur in temperate Asia, one in southern Europe (Poole 1989), and until now six species were recognized in North America (Lafontaine and Schmidt 2010). The current species concepts of Nearctic Raphia are essentially unchanged from those proposed by Smith (1908), although Raphia coloradensis was revised to the synonymy of Raphia frater by Schmidt and Anweiler (2010) and Raphia piazzi (Hill 1927), described after Smith’s work. Hence, over a century has elapsed since Smith’s comprehensive synopsis of the Nearctic species. In this study, we revise the taxonomy of the six North American Raphia taxa based on geographic variation in adult phenotype, genitalia morphology, mtDNA barcode variation, and larval host plant use.

Adult genitalia were prepared following the methods of Lafontaine (2004). Cleaned, stained genitalia were stored and examined in 30% ethanol, and slide-mounted in Euparal before being photographed using a Nikon D200 digital camera. Distribution maps were generated using SimpleMappr (http://www.simplemappr.net/). Tree distribution maps were adapted from USGS (2013).

We examined approximately 4000 specimens during the course of this study, primarily those of the CNC, EME, MEM, USNM, and UASM. Specimen repository abbreviations are as follows:

AMNH American Museum of Natural History, New York;

ANSP Academy of Natural Sciences, Philadelphia, PA;

BIO Biodiversity Institute of Ontario, Guelph, Ontario;

BMNH The Natural History Museum (statutorily British Museum [Natural History]), London;

CNC Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa;

CSU Colorado State University, Fort Collins;

EME Essig Museum of Entomology, University of California, Berkeley, California;

MEM Mississippi Entomological Museum, Mississippi State, MS;

UASM University of Alberta Strickland Museum, Edmonton, Alberta;

USNM National Museum of Natural History (formerly United States National Museum), Washington, D.C.

DNA extraction, PCR amplification, and sequencing of the COI barcode region were performed at the Canadian Centre for DNA Barcoding (CCDB) and followed standard protocols (Hebert et al. 2013; http://www.ccdb.ca/resources.php). Only sequence records greater than 500bp (range 500bp – 658bp) are included.

Morphology. Comparison of 20 genitalia dissections of each sex, representing all geographic entities, failed to reveal any diagnostic differences. The shape of the male valve apex and clasper varied slightly, but do so even within a single population. The shape of the inflated vesica, the most important diagnostic character in many noctuid species complexes, showed no discernible differences. Female genitalia were similarly conservative in variation. The European Raphia hybris Hübner, which is externally very similar to Raphia frater (and is in fact virtually indistinguishable from some Raphia frater coloradensis phenotypes), differs from Raphia frater in valve shape, vesica structure (including presence of spinules that are absent in Raphia frater) and shape and size of the corpus bursae. This indicates that Raphia genitalic morphology is not unusually homogeneous, where interspecific differences might be lacking.

The North American Raphia species have previously been delineated based on wing colour and pattern (Smith 1908), and geographic variation is considerable (Fig. 1). The most important forewing characters include ground colour, extent of medio-anal black shading, shape of the antemedial band, and the amount of fuscous shading of the hindwing. The colour of the prothoracic setae and overall size also vary.

Raphia has an extensive North American distribution, occupying virtually all biomes. Phenotypes are generally quite consistent regionally, but can appear drastically different in geographically disparate areas, which led early authors such as Smith (1908) to recognize multiple North American species. To assess phenotypic and mtDNA variation in these contact zones, we therefore attempted to locate and study specimens from key geographic regions where either two or more taxa would be expected to occur sympatrically or transition from one phenotype to another. The most comprehensive data were available for four such regions: a) the central Great Plains and b) the northeastern U.S., both where nominal ssp. frater interacts with ssp. abrupta; b) southern New Mexico where ssp. elbea meets ssp. coloradensis, and c) the Pacific Northwest / northern Rocky Mountains where sspp. coloradensis, frater and elbea meet (Figs 1, 3).

In the central Great Plains, a large series of over 60 specimens from northern Nebraska (Cherry Co.) is so variable that scarcely two individuals are alike, varying from the granular, dark grey forewing and white hindwing of ssp. frater, to the even, light grey forewing and slightly fuscous hindwing of Raphia frater abrupta; an intermediate specimen is shown in Fig. 1i. Some individuals show the blotchy black and grey pattern (with a contrasting black medio-anal shade) characteristic of Raphia frater coloradensis. A shorter series from Kansas (Riley Co.) falls within the variation of the Nebraska population. Interestingly, Smith (1908) remarked that Denver, Colorado specimens varied more towards ssp. abrupta than ssp. coloradensis. Single specimens from southeastern Montana and southwestern North Dakota have relatively pale forewings, like ssp. coloradensis, but some have fuscous hindwing shading and a dark prothoracic collar like abrupta. The zone of clinal variation may therefore be more extensive than the specimens from the few available sites in the northern Great Plains indicate, so further surveying in the region from southeastern Montana and southwestern North Dakota southward to western Nebraska / Kansas and eastern Colorado (particularly along major river corridors) would be helpful. Such a large transition zone appears to be the result of continuous, flat topography with a single, widespread Populus L. species (Populus deltoides Bartr.) that is utilized by both ssp. coloradensis (to the northwest) and ssp. abrupta (to the southeast). This transition zone corresponds closely with the suture zone first proposed by Remington (1968) and recently verified by others (Swenson 2010 and references therein).

The nature of the ssp. abrupta – frater interface is somewhat different in the Northeast, and is seemingly more influenced by topography and host plant distribution (Fig. 2); at least three Populus species occur regionally among topography ranging from coastal floodplains to the Appalachian Mountains. Specimens from the Pocono Mtns. of Pennsylvania are Raphia frater frater, whereas nearby central Maryland (Ann Arundel Co.) specimens (Fig. 1h) show transitional features in having a forewing pattern much like ssp. frater, but with a fuscous hindwing and a darker prothoracic collar characteristic of ssp. abrupta. Coastal Maryland (Montgomery Co.) specimens are typical Raphia frater abrupta (Fig. 1e). As discussed below, the transition zone between ssp. frater and ssp. abrupta seems to be mediated by habitat and host plant differences, with frater largely associated with aspen (Populus tremuloides Michx. and Populus grandidentata Michx.; Fig. 2) and ssp. abrupta with cottonwood (Populus deltoides; Fig. 2). Study of the populations on either side of the Ohio River is needed because frater occurs throughout Ohio (Rings et al. 1992), whereas the few northeastern Kentucky specimens that were examined are mostly like ssp. abrupta, but show some ssp. frater traits, including a mostly white hindwing. The Ohio River valley is an important suture zone between other biota, but the relative limits of Raphia frater frater and ssp. abrupta from the northern Appalachians eastward appear to be further south than recognized suture zones (Swenson 2010).

In southern New Mexico where elbea and coloradensis meet, elbea is known from the Mimbres Mountains (Grant Co.) in the southwest, with the nearest documented coloradensis locations 180 km to the northeast in the Rio Grande valley, and 300 km to the east in Eddy County (Fig. 1). The phenotypic transition between the two taxa is more abrupt in southern New Mexico than it is in the Great Basin where elbea imperceptibly transitions to the very pale Great Basin forms of coloradensis. A series from Twin Falls, Idaho, and some individuals from Leavenworth, Washington (Fig. 1p), are indistinguishable from Arizona elbea (Fig. 1q). Raphia frater elbea therefore grades into Raphia frater coloradensis in low-elevation habitats of the northern Great Basin. Specimens from the Eddy Co., New Mexico population are most like coloradensis, but some individuals are again indistinguishable from elbea. The mtDNA haplotypes associate this population with coloradensis (Fig. 3), and the available larval hosts are Populus angustifolia James and Populus deltoides (National Park Service 2014), but not Populus fremonti Wats. with which elbea is most often associated. Analysis of Raphia populations from the lower Rio Grande valley of New Mexico is desirable given the geographically intermediate position between elbea and coloradensis, and the presence of Populus fremonti (Fig. 3).

Populations at the edges of the Great Basin can be extremely variable, much like the situation between abrupta and frater in the north-central Great Plains. Series of specimens from Waterton Lakes, Alberta; Okanagan Valley, British Columbia; Baker County, Oregon; and Leavenworth, Washington range from the typical dark grey frater to pale yellowish coloradensis (Fig. 1). The Leavenworth population is remarkable in that it exhibits phenotypes ranging from frater to coloradensis (Fig. 1o) and elbea (Fig. 1p).

The geographic structure of California populations is not well documented; typical cinderella occurs from the San Francisco Bay area southward through the Central Valley to Los Angeles Co., but Raphia frater is apparently absent from southeastern California and the southern Sierra Nevada. Northern California (including the Sierra Nevada) specimens are most like Great Basin coloradensis but the transition from cinderella to this paler form is subtle, with Siskiyou Mountains material appearing intermediate. The Siskiyou Mountains are part of a northern California – southern Oregon suture zone also identified for other flora and fauna (see Swenson 2010 and references therein). Areas in central Texas where abrupta and piazzi meet, and eastern New Mexico/west Texas where the ranges of abrupta, coloradensis and elbea converge, remain unstudied.

Extreme phenotypic variation is therefore the modal geographic pattern at suture zones. In all cases where we examined suture zones between putative taxa, phenotype variation was moderate to extreme, and specimens could not consistently be assigned to existing taxonomic categories. Similarly, mtDNA variation patterns show no evidence of sympatric, reproductively isolated taxa, as discussed below.

Host plants. 72% of the 132 larval collections of Raphia frater from across Canada summarized by Prentice (1962) came from trembling aspen, 17% from other Populus species (Populus balsamifera L., Populus trichocarpa Torr. & Gray, Populus grandidentata, Populus ‘× canadensis’ (Alt.), Populus nigra L. var. italica du Roi), and 3% from Salix spp. Three records from white birch and one from alder (both Betulaceae) reported by Prentice are exceptional and possibly accidental. Wagner et al. (2011) consider Populus to be the main hosts, and question the validity of records from birch and alder. The closely-related European species, Raphia hybris, is restricted to Populus. Raphia larvae possess an unusually large number of proleg crochets (Beck 1996), a trait also seen in the Populus-feeding genus Ufeus Grote (Noctuidae: Noctuinae); this trait is postulated to be an adaptation to maintaining a grip on the leaves of aspen species (Lafontaine and Walsh 2013), which tremble even in slight breezes. Based on these data, Populus, and to a lesser extent Salix L. (both Salicaceae), are certainly the primary and probably the only larval host plants. Salix may be used only incidentally or in certain regions/habitats; the parallel paucity of Raphia and Populus occurrence in the central Appalachian region is notable (Fig. 2). Eight species of Populus occur in North America, divided into four sections: Leucoides Spach (Populus heterophylla L.), Aigeiros Duby (Populus deltoides, Populus fremonti), Tacamahaca Spach (Populus balsamifera, Populus trichocarpa, Populus angustifolia) and Populus L. (Populus grandidentata, Populus tremuloides). One additional species, Populus mexicana Wesmael of central Mexico, is the sole constituent of section Abaso Eckenwalder (Eckenwalder 1996).

Although it is reasonably certain that Raphia frater larvae are Salicaceae specialists, the geographic variation in host use and extent of specialization is not well understood. Nonetheless, it is possible to extrapolate broader host use patterns based on larval collections, host plant distributions and habitat associations. Below, we outline some potential scenarios of host use among Raphia frater subspecies.

Host plant records for Raphia frater frater (Prentice 1962) indicate that Populus tremuloides is probably the dominant, and certainly the most geographically widespread host (with the caveat that the high proportion of trembling aspen collections may simply reflect sampling bias). In eastern North America, the southern range limit of Raphia frater frater corresponds closely with the combined southern limits of the two aspen species (section Populus: Populus tremuloides and Populus grandidentata; Fig. 2). Raphia frater populations in riparian habitats of southern Alberta, where Populus tremuloides is scarce or absent, are associated with other Populus species that form a complex zone of hybridization and overlap among four species (Populus deltoides, Populus balsamifera, Populus trichocarpa, and Populus angustifolia) along the major river valleys (Brayshaw 1965, Floate 2004). In this region Raphia frater frater phenotypes transition to Raphia frater coloradensis (Fig. 3). The northernmost extent of Populus deltoides and Populus deltoides × balsamifera bybrids in the Red Deer River valley at about 52° latitude (Floate 2004) also coincides with the northernmost extent of Raphia frater coloradensis-like phenotypes; north of there where Populus tremuloides is the dominant species of the Aspen Parkland ecoregion, only pure Raphia frater frater phenotypes occur. Similarly, transitional frater-coloradensis populations occur in southwestern Alberta, southern British Columbia and central Washington at the range edges of Populus tremuloides, where Populus trichocarpa becomes the dominant Populus (Fig. 3). In northern Labrador, Raphia frater frater is at its northeastern range limit (not shown), occurring beyond the range of Populus; Salix species are the presumed hosts.

Throughout most of the range of Raphia frater abrupta, Populus deltoides is the only Populus species present. Swamp cottonwood (Populus heterophylla) has a small eastern North American range, occurring primarily along the Mississippi and Ohio River valleys and along the Atlantic seaboard (see e.g., Sibley 2009, USGS 2013), so this may serve as a host in some areas. The hosts for the southwest Texas taxon Raphia frater piazzi are unknown, and may constitute willows rather than Populus, the latter being rare or absent where Raphia frater piazzi occurs (Fig. 3).

In the Pacific Northwest, Raphia frater frater is associated with Populus tremuloides in northern Washington and British Columbia, with Raphia frater coloradensis of dry, low-elevation habitats associated with Populus trichocarpa (L. Crabo, pers. comm.). Crumb (1956) documented a larval collection from the latter species in south-central Washington. Throughout most of the Pacific Northwest, the only Populus species are Populus tremuloides at upper elevations, and Populus trichocarpa in low-elevation riparian habitats and drier soils in moist regions (Fig. 3). A third species, Populus angustifolia, occurs locally in the eastern parts of the Pacific Northwest, with habitats similar to Populus trichocarpa. There are no specific host records for the Californian Raphia frater cinderella, with both Populus trichocarpa and Populus fremonti being the most likely hosts. Along the east slopes of the Oregon Cascade Ranges to the north, Raphia frater coloradensis associates with Populus trichocarpa (L. Crabo, pers. comm.) Dyar (1894) cites “poplar” as a foodplant for larvae from Yosemite. Subspecies coloradensis in the Sierra Nevada, to the east of the central Californian range of cinderella, is associated with Populus tremuloides.

Arizona populations of Raphia frater elbea feed on Populus fremonti (Crumb 1956; D. Wagner, pers. comm.), and again this is the only available Populus in much of the range of ssp. elbea, excepting the higher elevations in the mountain ranges of central Arizona where Populus tremuloides and Populus angustifolia occur (Fig. 3). A population of Raphia frater coloradensis in the Rio Grande valley of central New Mexico is also associated with riparian Populus fremonti (Fig. 3). Aspen-associated, Raphia frater frater-like populations may occur at high elevations in Arizona, similar to the situation in Colorado, but this has not been documented. This raises the interesting possibility that frater occurs at higher elevations, with elbea occurring in the low-elevation floodplain. Surveying at the uppermost elevations of the Chiricahua and Santa Catalina Mountains of southeastern Arizona where aspen occurs have so far not yielded Raphia (BCS unpubl. data), but the more expansive range of Populus tremuloides along, for example, the Mogollon Rim is poorly surveyed.

In summary, larval host plant associations of Raphia frater populations shows some broad congruencies between subspecies and Populus species distributions, but with limited evidence for high host fidelity: range edges of Raphia frater subspecies generally do not closely follow those of the various Populus hosts, suggesting that Populus availability rather than high host fidelity may be the limiting factor to Raphia distribution, and that climatic and topographic effects have a greater selective influence that does host plant specialization. To what extent these congruencies reflect common co-evolutionary trajectories, and what factors drive intraspecific divergence, would be a fascinating and fruitful area of study.

Molecular variation. The deepest splits in mtDNA barcode variation (alluded to previously in Lafontaine and Schmidt 2010) segregate North American Raphia into four groups, but only one of these is private to a recognizable taxon (Raphia frater elbea). None of the remaining subspecies exhibited discrete haplotypic variation. Based on analysis of 192 specimens from localities across the range of Raphia frater representing all subspecies (Suppl. material 1), haplotypes segregated into five groups: 1) a large group from across most of the eastern, northern and central range portions that includes Raphia frater frater, Raphia frater coloradensis, Raphia frater abrupta and Raphia frater piazzi, varying by up to ~1.3% (Fig. 4); 2) a discrete group closest to haplogroup 1, consisting of two Raphia frater piazzi specimens (Fig. 4); 3) a divergent group of geographically disparate samples of Eastern Raphia frater frater (Ontario, Manitoba) and Californian Raphia frater cinderella specimens, differing by a minimum of ~2.2% from all other groups (Fig. 4); 4) a group private to Raphia frater elbea (Arizona, New Mexico, Utah) with a minimum ~1.0% divergence; 5) A group of Californian Raphia frater cinderella with a minimum divergence of ~1.8% from all other haplotypes.

The combination of California and eastern Canada samples in haplogroup 3 to the exclusion of all others was quite unexpected, given the geographic structuring of other haplogroups. Two haplogroup-3 populations (Bird Hill, Manitoba; Bruce Peninsula, Ontario) also exhibited group 1 haplotypes (Fig. 4), the only sampled populations to yield more than one haplogroup. Representative specimens from these sites were of the same phenotype and from the same sampling event. This haplogroup could therefore be a retained ancestral mtDNA polymorphism, or indicative of Wolbachia-induced mtDNA lineage sorting similar to that documented by Kodandaramaiah et al. (2013). Determining the underlying cause of this interesting variation will require study using nuclear gene markers and Wolbachia assays.

Considering the general lack of taxonomic resolution of North American Raphia in the barcode sequence, and comparing divergences among Palaearctic Raphia as a metric of mean species divergences within the genus, mtDNA variation is most parsimonious with a geographically structured, single-species interpretation. The contrast between often considerably different adult phenotypes and lack of significant mtDNA and morphological differentiation may reflect strong regional selection on bark-cryptic wing patterns, which in turn is dependent on dominant host trees that vary according to regional host preferences.

The North American Raphia populations exhibit considerable geographic variation in phenotype, previously segregated into six species. Despite these geographically structured phenotypic differences, diagnostic morphological differences in genitalia and larvae are not evident. Scrutiny of geographic contact zones between putative taxa revealed populations with extensive phenotypic and conservative molecular variation, rather than bimodal phenotypic variation coupled with deep molecular divergences that would be expected for sympatric, reproductively isolated taxa. Raphia frater larvae are not highly restricted to a host species or genus, but do specialize on Populus and Salix, with a pattern of regional host availability and possibly also preference. Differences in host plant suitability among the various species of Salicaceae remain unstudied. Assessment of morphology, mtDNA variation, and biogeography therefore leads us to conclude that the geographic segregates of North American Raphia are best treated as subspecies of a single species. The regional adaptation to habitats representing nearly all North American biomes, combined with relatively discrete geographic ranges of unique adult phenotypes, suggest a pattern of young or incipient species in the Raphia frater group.

The taxonomy and biogeography of the North American Raphia populations is a complex interplay between topography, host plant use, phenotypic variation and evolutionary history. This study is only the first attempt at a better understanding of this interesting group. Many questions remain unanswered: what are the exact geospatial and host plant patterns of the contact zone between Raphia frater abrupta and Raphia frater frater? Is there geographic overlap with altitudinal segregation in the West between aspen-feeding frater and cottonwood feeding elbea? Does the mtDNA haplogroup 3 represent Wolbachia infection? Do the lower Rio Grande / Edwards Plateau piazzi populations grade into abrupta? Raphia would provide a fertile area of study in understanding large-scale patterns of host plant use and biogeography of a widely distributed continental Lepidopteran.