Viola kauaensis var. hosakae (Violaceae), a new variety of endemic Hawaiian violet

Abstract The Hawaiian endemic Viola kauaensis A. Gray has a broad distribution in bogs of Kaua`i and a limited distribution on mesic ridges in the Ko`olau Mountains of O`ahu. Based on differences in scale, the O`ahu populations of Viola kauaensis had previously been described as a distinct taxon. The taxonomic status of the O`ahu populations was reevaluated through a morphometric analysis of all varieties of Viola kauaensis and the morphologically similar Viola vanroyenii. Morphological features of historic and freshly collected specimens of all varieties of Viola kauaensis were analyzed with a principal components analysis. Populations from O`ahu represent a distinct cluster that slightly overlaps with Viola kauaensis var. kauaensis. Lamina width, apex angle, and base angles contribute to the separation of the O`ahu populations from other varieties of Viola kauaensis. Due to differences in scale, the O`ahu populations are described as Viola kauaensis var. hosakae, a new critically endangered taxon.


Introduction
Viola kauaensis A. Gray is one of nine species of the monophyletic Hawaiian violets (Violaceae) Sytsma 2000, Havran et al. 2009). Th e species grows from a creeping rhizome and is notable among endemic Hawaiian Viola in being both herbaceous and bears cleistogamous fl owers (Wagner et al. 1999). Two varieties of V. kauaensis are recognized: V. kauaensis var. kauaensis possesses rotund to cordate leaves and is distributed primarily in high elevation bogs and cloud forest margins in central to northwestern Kaua`i (Wagner et al. 1999); Viola kauaensis var. wahiawaensis Forbes is distributed in the Kanele (Wahiawa) Bog and nearby ridges north of the town of Hanapepe, Kaua`i (Forbes 1920, Wagner et al. 1999. Viola k. wahiawaensis can be diff erentiated from V. k. var. kauaensis by cuneate leaf bases (Forbes 1920, Wagner et al. 1999. While V. k. var. kauaensis is locally abundant, V. k. var. wahiawaensis is a federally listed endangered taxon. Additional populations of V. kauaensis are located in the Ko`olau Range on the neighboring Hawaiian Island of O`ahu. Individuals in the O`ahu populations resemble V. k. var. kauaensis but possess smaller leaves, stipules, fl owers, and fruits (St. John 1989). Viola kauaensis is rare on O`ahu and is distributed on sloping exposed or mossy ground, not in open bogs or forest margins like on Kaua`i. No evidence has been found that the populations on O`ahu produce cleistogamous fl owers. Fosberg and Hosaka (1938) mention that "the specimens from O`ahu correspond very well with the dwarf form from the bogs" of Kaua`i. Due to diff erences in scale, the individuals in the O`ahu populations were named Viola hosakae St. John by St. John (1989) in a systematic treatment of all Hawaiian Viola. Th e O`ahu specimens of V. kauaensis were not available for study during the drafting of Manual of the Vascular Plants of Hawai`i (Wagner et al. 1999) and were therefore not treated in that publication. In Th e Flora of the Hawaiian Islands website, V. hosakae is placed in synonymy with V. k. var. kauaensis but W. Wagner noted: "probably this should be treated as a third taxon of V. kauaensis". St. John (1989) named one other violet that is morphologically similar to Viola kauaensis. Viola vanroyenii St. John represents a population of small herbaceous violets endemic to the summit area of Mt. Wai`ale`ale on Kaua`i. One collection was made from the summit by van Royen and Perlman in 1977 (P. van Royen 11733 [BISH]). Th e population is represented by one herbarium sheet at BISH containing 14 individual plants and several fragments. Th e species is distributed within the range of V. k. var. kauaensis on Kaua`i and has yet to be rediscovered (Ken Wood and Steve Perlman, personal communication).
In the current study, the taxonomic status of O`ahu populations of V. kauaensis was reevaluated through an analysis of vegetative and reproductive traits of all varieties of V. kauaensis and of V. vanroyenii. We asked three questions: (1) Is variation in morphological traits discontinuous between interisland populations of Viola kauaensis?; (2) Do the O`ahu populations of V. kauaensis produce cleistogamous fl owers?; and (3) Should the O`ahu populations of V. kauaensis be treated as a distinct taxon?

Field collection
Viola kauaensis has been documented from several sites on O`ahu. Th e fi rst recorded collection of the species in 1938 was recorded as "Ko`olau Range, divide between head of Kawainui and Kaipapau Gulches" (E.Y. Hosaka 2,504 [BISH]). Th e site has not been relocated since the original collection. Two additional populations are located in the Poamoho region of the Ko`olau Mountains (due to the rarity of the species, the population locations are referred here as sites A and B). Both locations were visited in May 2013 to make new collections and assess the size of populations.
Site A is located near the Poamoho Trail and contains four small subpopulations. Because the species is considered locally threatened only two whole individuals were collected. One additional fl ower was collected and preserved in 70% ethanol for dissection. Th e fragmented nature of the populations and their position on nearly vertical cliff faces (which appears typical for individuals on O`ahu) prevented a random assessment of individuals from the site. To obtain a measure of the size of individuals at Site A, the length and width of the largest lamina of several individuals was measured. A 6 m transect was run parallel to the summit of one ridge. A 2 m tape was extended down the slope of the cliff every 1 m. Individuals easily accessed within 50 cm of the tape were measured.
Site B is located along the Ko`olau Summit Trail. Th e population was discovered in 1986 by John Obata. Information on the population was shared with Clyde Imada of the Bishop Museum Herbarium (personal communication). Th e site was revisited by Clyde Imada in 1995 who did not observe any Viola at that time. In May 2013, Site B was revisited to survey for Viola kauaensis. No individuals of V. kauaensis were rediscovered at Site B.
Label data from the type of V. vanroyenii collected in 1977 (van Royen 11733) indicates that all specimens of V. vanroyenii were collected from the "summit area of Mt. Wai`ale`ale". In November 2012, the summit of Mt. Wai`ale`ale was visited by Kyle Kagimoto (Th e Nature Conservancy of Hawai`i). Five samples of V. kauaensis were made from the summit area and are included in the current study.

Measurements
All specimens representing varieties of V. kauaensis and V. vanroyenii on deposit at BISH and DUKE herbaria were analyzed. Digital scans of O`ahu specimens of V. kauaensis from PTBG were examined. Th e type specimens of V. k. var. wahiawaensis, V. vanroyenii, and V. hosakae were analyzed at BISH. A digital scan of the type of V. kauaensis from US was analyzed. Five specimens of V. kauaensis collected by Kyle Kagimoto were deposited at CAU. Morphological variables measured include: length and width of the leaf lamina, cauline stipules, rhizome stipules, and sepals; length of petioles and capsule valves; and apex and base angles of the leaf lamina. Very few specimens of V. kauaensis from O`ahu possessed intact or fully developed petals. Th erefore we restricted our fl oral measurements to sepal characters only. Th e largest leaf on each specimen was chosen for measurements of foliar characteristics. If a leaf was folded, damaged, or wrinkled to the extent that it could not be determined if it represented the largest leaf, the next largest leaf was chosen for measurement. All size measurements were made to the nearest 0.5 mm. Apex angle was measured as the angle of two rays running along the margins of the leaf tip with the vertex placed just above (at or within 1 mm) of the leaf tip. Base angle was measured as the angle of two rays running along the base of the lamia with the vertex placed just below (at or within 1 mm) of the tapered base of the lamina (Ellis et al. 2009). Some specimens of V. k. var. kauaensis possessed cordate leaf bases. In these cases, base angle was measured as the angle of two rays running along the inner margins of the left and right portions of the reniform base with a vertex placed at the insertion point of the lamina (Ellis et al. 2009). For one folded leaf of V. k. var. wahiawaensis, base angle was estimated based on one half of a folded leaf.
Only one sheet of V. vanroyenii exists at BISH. Th e sheet contains 14 individual stems and several fragments. We attempted to measure as many entire samples from this sheet as possible to obtain a robust estimate of morphological variation in the taxon. Only three individuals on the sheet possessed all traits required for a principal components analysis.
Multiple individuals were measured from herbarium sheets when it appeared that vegetative and reproductive structures were attached to separate rhizomes. Digital images of herbarium specimens were used where possible. ImageJ software (Rasband 2012) was used to analyze digital images.

Analyses
A principal component analysis (PCA) was used to investigate the morphological variation between interisland populations and varieties of V. kauaensis. Th e PCA was conducted with varimax rotation on untransformed data. Many herbarium specimens contained samples with degraded, fragmented, missing, or not-otherwise obvious characteristics. Th erefore, only specimens that had complete measurements for lamina length, lamina width, petiole length, apex angle, base angle, cauline stipule length, and cauline stipule width were included in the analysis. Th e 49 samples incorporated into the PCA are listed in Table 1. In accordance with the Kaiser rule, principal component (PC) loadings with eigenvalues above 1.0 were retained for further analysis. Analyses were conducted in R version 2.15.1 (R Core Team 2012).

Floral morphology
Only specimens of V. k. var. kauaensis from Kaua`i and V. k. var. wahiawaensis possessed open and mature fl owers suitable for measuring petals. All specimens of V. vanroyenii and V. kauaensis from O`ahu possessed either cleistogamous fl owers or fl owers without fully developed petals. St. John (1989) included a three dimensional sketch of a chasmogamous fl ower and sketches of dissected fl oral organs in his description of the type of V. hosakae. Due to the degraded nature of the petals in a fragment envelope of the type of V. hosakae at BISH we were unable to reassess size and shape of fl oral organs from O`ahu. St. John's (1989) measurements of fl oral organs from V. hosakae are referenced in discussions of petal size.  Although no V. kauaensis individuals with chasmogamous fl owers were observed during the 2013 surveys on O`ahu, several unopened fl owers were observed. In the fi eld, it was not obvious if the fl owers represented unopened chasmogamous fl owers or fully developed cleistogamous fl owers. One fl ower was collected and preserved in 70% ethanol (multiple fl owers were not collected to reduce detrimental impact on the small population). Th e fl ower was rehydrated in distilled water prior to dissection. Floral organs were removed and attached to an archival slide. Th e size and shape of fl oral organs were compared to Skottsberg's (1940) illustrations of cleistogamous and chasmogamous fl oral organs from Kaua`i individuals of V. kauaensis.

Field measurements
Site A contained approximately 70 individuals scattered throughout four isolated patches. Th e violets grow from a layer of exposed moss on heavily sloped areas ( Figure 1). All violets in the area had a small stature, less than 5 cm in height above the moss layer. No conspicuous chasmogamous fl owers were observed. Lamina dimensions in the fi eld ranged from 2-16 mm in length to 2-15 mm in width. Th e average leaf lengths and widths were 11 and 11.5 mm, respectively. Despite a thorough search, no individuals of V. kauaensis were observed at site B. Th e PCA yielded three principal components (PC) with a cumulative proportion of 0.8831 (Table 3). Th e fi rst two PCs possessed eigenvalues greater than 1.0 and were retained for the construction of a biplot (Figure 2). Th e biplot depicts overlap between V. k. var. wahiawaensis and V. k var. kauaensis. Th ese two varieties are primarily separated along PC2, controlled by apex angle and base angle ( Table 2). Individuals of V. kauaensis from O`ahu cluster together but overlap slightly with individuals of V. kauaensis from Kaua`i. Viola vanroyenii completely overlaps with individuals of V. kauaensis from O`ahu. Th e O`ahu and Kaua`i populations of V. kauaensis are primarily separated along PC1, controlled by lamina width (Table 3).

Floral morphology
Removal of sepals from the preserved fl ower (V. kauaensis from O`ahu) indicated the presence of several withered petals and only two stamens with anthers. Th e stamens contained an elongated fi lament with anthers at their tip. Th e anther from one of the stamens was in direct contact with the stigmatic surface of the pistil. Th e style was also relatively short and curved towards the anther. All of these observations were consi stent with Skottsberg's (1940) illustrations of cleistogamous fl oral organs in V. kauaensis specimens from Kaua`i.

Discussion
Herbarium and fi eld data suggest that the herbaceous Viola on O`ahu occupy a much more limited range of morphological variation than V. k. var. kauaensis. Individuals in the O`ahu population, like those individuals on Kaua`i, also produce cleistogamous fl owers. Th e individuals on O`ahu demonstrate a fi xed range of variation in the size of leaves, petioles, cauline stipules, and fl owers. Although not evidenced in the PCA by contribution of base angle along PC1, individuals on O`ahu do not possess the extreme reniform leaf bases often observed in V. k. var. kauaensis. Th is may just be a trait that is exaggerated in larger leaves. Due to these variations, the O`ahu individuals are best treated at a distinct infraspecifi c rank within V. kauaensis: V. kauaensis var. hosakae.
Recently collected specimens on O`ahu represent a much smaller range of size than those individuals collected early in the 20 th century. Leaves of recently collected materials are considerably smaller than those in the type (Figure 3) and represent those individuals more distinct from V. k var. kauaensis in the PCA biplot. Viola kauaensis has been collected from multiple sites on O`ahu, but is now known from just one population. Th e variety may have existed across the Ko`olau Mountains in a wide range of sizes, but now persists as a solitary population in the smaller extreme of leaf size. Th e reduction in range size may be associated with interaction with non-native species. Th e invasive grass Axonopus fi ssifolius (Raddi) Kuhlm has had a negative impact to summit plants on O`ahu and is found growing alongside V. k. var. hosakae. Th e action of ungulates along the summit area would also detrimentally impact the survivorship of the variety.
Th e varieties of V. kauaensis on Kaua`i and O`ahu occupy diff erent habitats. On Kaua`i V. k. var. kauaensis is distributed in the open bog and cloud forest margins of the high-elevation Alakai Swamp. In the bog environments, the species is usually distributed in hummocks of Metrosideros polymorpha Gaud., mosses, and lichen, while in bog margins the species can be found growing terrestrially or epiphytically in pockets of moss on tree stems. On O`ahu, the one population of V. k. var. hosakae contains at least four smaller subpopulations of 4-30 individuals scattered over an area of about 50 m 2 . Each subpopulation is distributed on a moderate to steeply sloping surface with individuals growing directly out of unsaturated exposed soil or from a thin layer of moss (Figure 1). Th is microhabitat description diff ers greatly from the typical habitat of V. k. var. kauaensis on Kaua`i (Wagner et al. 1999), especially with regard to the slope. Havran et al. (2009) and Ballard (2000) included the O`ahu herbaceous violets in their phylogenies of the endemic Hawaiian Viola. In both studies of the Internal Transcribed Spacer (ITS) sequences, the O`ahu populations grouped closely with V. k. var. kauaensis. Neither study incorporated material from V. k. var. wahiawaensis. Th e O`ahu individual possessed four diff erences in the ITS sequence regions compared with the Kaua`i material. Th e variation is one of the largest seen when comparing interisland populations of conspecifi cs in the wet clade of Hawaiian violets. Viola k. var. hosakae likely diverged from V. k. var. kauaensis following an interisland dispersal event from Kaua`i to O`ahu. While it is likely that V. k. var. hosakae may have been derived through allopatric speciation, the relationship between V. kauaensis and V. vanroyenii is less clear. Viola vanroyenii falls within the range of morphological variation as V. k. var. hosakae, but outside the range of variation of V. k. var. kauaensis along PC1. Field observations by Steve Perlman (personal communication) indicate that V. vanroyenii is sympatric with V. k. kauaensis on Kaua`i. Viola vanroyenii may represent V. k. var. kauaensis at the smaller extreme of its morphological variation, possibly as a result of harsh conditions at the summit area of Mt Waiale`ale. If more individuals are ever found, this relationship should be reevaluated. Description. Rhizomatous herb, rhizome creeping rhizome stipules 1.5-3.0 mm long, 1-2 mm wide, often overlapping and scaly in appearance; vertical stems produced from rhizome, internodes on vertical stem longer than on rhizome, stipules 2.0-5.0 mm long, 1.0-2.5 mm wide. Flowers solitary on terminal peduncle, fl ower subtended by opposite pair of small linear bracts on peduncles. Chasmogamous fl ower characteristics as in St. John (1989): dorsal sepal 5 × 1.4 mm, elliptic; lateral sepal 4.5 × 1.4 mm, obovate elliptic; ventral sepal 5.6 × 1.4 mm, lance elliptic; dorsal petals 15 × 3.3 mm, with a 4 mm claw and an elliptic blade; lateral petals 14 × 2.6 mm, with a broad 4 mm claw and an elliptic blade; ventral petal 16 mm long, with a curved 6 mm channeled claw, and an elliptic blade that is 5 mm wide; dorsal stamen 3.9 mm long, fi lament 0.5 mm long, stout, oblique, anther 2.5 mm long, narrowly obovoid ellipsoid, sterile tip 1.3 mm long, ovate; lateral stamen 3.9 mm long, fi lament 0.5 mm long and broad, anther 2.3 mm long, narrowly cuneoid, sterile tip 1.3 mm long, ovate, acute; ventral stamen 3.6 mm long, fi lament 0.5 mm long and wide, anther 2.3 mm long oblanceoloid, sterile tip 1.5 mm long, lanceolate, nectary 1.5 mm high, 0.8 mm wide, arcuate oblong, basal; pistil 2.8 mm long; style 1 mm long; stigma discoid, divergent at 45°; chasmogamous fl owers not seen (see methods). Cleistogamous fl owers with linear sepals 5, green, 5-6 mm long, 1 mm wide, bases auriculate, apices acuminate, enclosing all other fl oral organs; petals 5 or fewer, up to 3 mm long, 1 mm wide, white, with withered appearance; stamens 2, 1.5 mm long, fi lament 1 mm long, anthers 0.5 mm long and at end of fi lament, anther in direct contact with stigmatic surface of pistil; pistil 2 mm long, ovary 1.5 mm long, style 0.5 mm long, curved at approximately 180˚ towards ovary. Fruit a capsule, capsule valves 7-9 mm long.

Viola kauaensis
Distribution var. hosakae appears very rare on O`ahu. Despite frequent and thorough conservation work by multiple organizations in the summit area of the Ko`olau Mountains, only one population of the variety is known to exist. Th e population is threatened by grazing ungulates. In addition, island tropical montane environments, like the ones harboring V. k. var. hosakae, are incredibly susceptible to global climate change (Loope and Giambelluca 1998).
Viola k. var. hosakae is best classifi ed as Critically Endangered (CR) according to the IUCN Red List Criteria as it meets the following criteria: B. Area of occupancy less than 10 km 2 , number of populations = 1, and continuing decline inferred from extent of occurrence and area of occupancy as indicated from herbarium records and personal communication; C. Number of mature individuals less than 250 and an estimated continuing decline (C2) with less than 50 mature individuals in each subpopulation (C2i).
Th e Plant Extinction Prevention Program (PEPP) branch on O`ahu will work to preserve this taxon on by collecting and germinating seeds when possible. Eff orts are underway to enclose the population within an ungulate fence by the end of 2014. An additional population can be started with propagules from the extant population. Th e cleistogamous reproduction of the variety should help to facilitate seed production in the absence of pollinators at a new location.