Viola kauaensis has been documented from several sites on O`ahu. The first 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]). The 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 flower was collected and preserved in 70% ethanol for dissection. The 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. The population was discovered in 1986 by John Obata. Information on the population was shared with Clyde Imada of the Bishop Museum Herbarium (personal communication). The 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 Viola kauaensis were rediscovered at Site B.

Label data from the type of Viola vanroyenii collected in 1977 (van Royen 11733) indicates that all specimens of Viola 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 (The Nature Conservancy of Hawai`i). Five samples of Viola kauaensis were made from the summit area and are included in the current study.

All specimens representing varieties of Viola kauaensis and Viola vanroyenii on deposit at BISH and DUKE herbaria were analyzed. Digital scans of O`ahu specimens of Viola kauaensis from PTBG were examined. The type specimens of Viola kauaensis var. wahiawaensis, Viola vanroyenii, and Viola hosakae were analyzed at BISH. A digital scan of the type of Viola kauaensis from US was analyzed. Five specimens of Viola 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 Viola kauaensis from O`ahu possessed intact or fully developed petals. Therefore we restricted our floral measurements to sepal characters only. The 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 Viola kauaensis 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 Viola kauaensis var. wahiawaensis, base angle was estimated based on one half of a folded leaf.

Only one sheet of Viola vanroyenii exists at BISH. The 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.

A principal component analysis (PCA) was used to investigate the morphological variation between interisland populations and varieties of Viola kauaensis. The PCA was conducted with varimax rotation on untransformed data. Many herbarium specimens contained samples with degraded, fragmented, missing, or not-otherwise obvious characteristics. Therefore, 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. The 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).

Only specimens of Viola kauaensis var. kauaensis from Kaua`i and Viola kauaensis var. wahiawaensis possessed open and mature flowers suitable for measuring petals. All specimens of Viola vanroyenii and Viola kauaensis from O`ahu possessed either cleistogamous flowers or flowers without fully developed petals. St. John (1989) included a three dimensional sketch of a chasmogamous flower and sketches of dissected floral organs in his description of the type of Viola hosakae. Due to the degraded nature of the petals in a fragment envelope of the type of Viola hosakae at BISH we were unable to reassess size and shape of floral organs from O`ahu. St. John’s (1989) measurements of floral organs from Viola hosakae are referenced in discussions of petal size.

Although no Viola kauaensis individuals with chasmogamous flowers were observed during the 2013 surveys on O`ahu, several unopened flowers were observed. In the field, it was not obvious if the flowers represented unopened chasmogamous flowers or fully developed cleistogamous flowers. One flower was collected and preserved in 70% ethanol (multiple flowers were not collected to reduce detrimental impact on the small population). The flower was rehydrated in distilled water prior to dissection. Floral organs were removed and attached to an archival slide. The size and shape of floral organs were compared to Skottsberg’s (1940) illustrations of cleistogamous and chasmogamous floral organs from Kaua`i individuals of Viola kauaensis.

Site A contained approximately 70 individuals scattered throughout four isolated patches. The 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 flowers were observed. Lamina dimensions in the field ranged from 2–16 mm in length to 2–15 mm in width. The average leaf lengths and widths were 11 and 11.5 mm, respectively. Despite a thorough search, no individuals of Viola kauaensis were observed at site B.

Data from herbarium specimens show Viola kauaensis populations from O`ahu possessed consistently smaller mean values of morphological and reproductive traits when compared to populations on Kaua`i (Table 2). Minimum ranges of morphological values from Kaua`i overlap with maximum values from the O`ahu individuals.

The PCA yielded three principal components (PC) with a cumulative proportion of 0.8831 (Table 3). The first two PCs possessed eigenvalues greater than 1.0 and were retained for the construction of a biplot (Figure 2). The biplot depicts overlap between Viola kauaensis var. wahiawaensis and Viola kauaensisvar. kauaensis. These two varieties are primarily separated along PC2, controlled by apex angle and base angle (Table 2). Individuals of Viola kauaensis from O`ahu cluster together but overlap slightly with individuals of Viola kauaensis from Kaua`i. Viola vanroyenii completely overlaps with individuals of Viola kauaensis from O`ahu. The O`ahu and Kaua`i populations of Viola kauaensis are primarily separated along PC1, controlled by lamina width (Table 3).

Removal of sepals from the preserved flower (Viola kauaensis from O`ahu) indicated the presence of several withered petals and only two stamens with anthers. The stamens contained an elongated filament with anthers at their tip. The anther from one of the stamens was in direct contact with the stigmatic surface of the pistil. The style was also relatively short and curved towards the anther. All of these observations were consistent with Skottsberg’s (1940) illustrations of cleistogamous floral organs in Viola kauaensis specimens from Kaua`i.