RichardR. Halse,4535 N.W Big Oak place,No. 3, Corvallis,Oregon 97330 Bruce A. Rottink,' and RichardMishaga, CH2M Hilr,rnc. 2020SW FourthAvenue, Portland, Oreqon g720.1

Studiesin SidalceaTaxonomy

Abstract

The-objectives of this study were to inyestigate taxonomic relationships among the four species of growing in Oregon,s Willamerte Valley, and among various populations of Sid,alceaneioniona.lhese relaiionships were u..""s..d b'y exa-rning with the scanning electron microscope, and performing principal component analysis(PCAfon gross mo.phologi"uii.uru.". of the . Pollen morphology was of limited use in making intra- and inter-specific comparisons. Chro-o.o-e"number was determined for six populations of S. nelsoniana; it was identlcal for all populaiions (n = to). pCA was useful in segregatrng the four Sidalcea species. However, a PCA of 73 specimens of S. neLsoniina revealed no distinct sub-taxa; this i.flrrira'o, is useful in making management decisions for this Category 2 candidate species.

Introduction existenceof S. nelsonianain thesetwo different habitats has led to speculationthat there may Sidalcea is comprised of herbaceousannuals and be two separatetaxa. This paper also examinesthis perennials native to western North America hypothesis. (Roush 1931,Hitchcock 1957).The four species The proposed found in the Willamerte Valley of Oregln, S. constructionof a water supply reservoir at Walker nelsoniana Piper (Nelson's checker-mallow),S. Flat in the Coast Range, west of Carlton, would inundate cusickii Piper (Cusick's checker-mallow), S. an area which cur- rently supports a large population campestris Greene (meadow sidalcea). and S. of S. nel- soniana (Glad et al. 1987). Determining airgata Howell (rose checker-mallow), are all the tax- onomic status of the S. nelsonianaat perennials that bloom in late spring to mid- Walker Flat would help federal agenciesassess the summer and are characterizedby a gynodioeci- signifi- cance of managing this population. ous breeding system.In a gynodioeciousspecies individual plants are either pistillate (the flowers Materialsand Methods are male-sterile)or hermaphroditic (the flowers are perfect). Often morphological differences, Relationshipsamong the four speciesof Sidalceo, such as overall size or flower size (Willson and within S. nelsoniana,were determined by ex- 1983),can be found between these two types of amination of pollen with a scanning eleciron individuals. The existence of these two different microscope (SEM), and principal Jomponent morphological types within a specieshas resulted analysis(PCA) of selectedmorphological char- in frequent misidentification of these species. acteristics. In addition, chromosome counts were Therefore, the primary goal of this study was to made for six S. nelsoniana populations, two from clarify species relationships by examining se- the Coast Range and four from the Willamette lected taxonomic and morphologic characteristics Valley. of these species. Air dried pollen collectedfrom the four spe- ciesof Sidalceawas dispersedon l5 mm diameter The of S. nelsoniana, a western coverslips coated with a thin adhesive film Oregon endemic, has been a concern to federal (Mikrostick). Coverslipswere attached to alumi agenciesas it is a candidate speciesfor federal num planchets using conductive silver paint. listing as a Category 2 species(U.S. Fish and After mounting, spqcimenswere coatedwith ap- Wildlife Service 1985).It is found in the '|50 northern proximately A of 60:40 weight p"r..nt CoastRange in both the Nestucca River and the Au/Pd alloy in a Varian VE-10 vacuum evapo- Wilson River drainages, and in the Willamette rator at a vacuumof I x l0-s Torr. Examination Valley from Benton and Linn Counties north to was made using an AMRAY 1000 A SEM, oper- Washington (Halse County and Glad l986). The ated at l0 or 20 kV, at rhe Electron Microscone Facility, Departmenr of and paih- rCurrent Plant Address:l4 Touchstone,Lake 0swego, 0regon 92035 ology, Oregon State University. Images were

154 NorthwestScience, Vol. 63, No. 4, 1989 recorded on Polaroid Type 55 positive/negative TABLE 1. Definition of morphologicalparameters used in 4x5formatfilm. Sidalceaprincipal componentanalyses.

For chromosome determination, floral buds Abbreviation Definition Numeric Code from six populations of S. nelsonianawere col- lected in the field and fixed in a modified Car- BASTPU basal stem I = none noy's solution (4 chloroform: 3 ethanol: I glacial pubescence 2 = simple = acetic acid, v/v/v). Acetocarmine squashes of 3 forked or stellate pollen mother cells were obtained by using the PETCOL petal color I = white or pink = technique of Snow (1963). 2 red, lavender or purple

The morphological measurements for this ADPETLG adjusted petal millimeters study were obtained from l3l herbarium speci- lcnoth mens of the four species of Sidalcea borrowed ADCATTG adjusted calyx millimeters from the herbaria at Oregon State University, the length University of Oregon, Washington State Univer- CAtCOt calyx color I = green sity, the University of Washington, Willamette 2 = green with some University, and the United States Fish and Wild- purple = life Service,Boise, Idaho. Of thesespecimens 73 J purple were of S. nelsoniana,22 of S. campestris,2l of S. uirgata and l5 of S. cusickii. Specimens were from throughout the species'distribution in the species on the length of the petals and calyces. Willamette Valley. A list of the specimens,and Therefore, for the PCA, the length of the petals their site localities,used in the PCA is available and calyces were adjusted for sex-related flower from the senior author on request. differences. For both petals and calyces,this ad- justment was made separatelyfor each species The five morphological parameterslisted in by adding the difference between the mean Table I were measured on all l3l herbarium Iength of the perfect-flowered specimensand the specimens.Only those specimenswhich had all mean length of the pistillate-flowered specimens five morphological parameterswere used in the to each of the pistillate-flowered individuals. PCA. These parameters nere selected for the Following this adjustment, the mean length of PCA becausethey are considered to be impor- the pistillate-flowered individuals was equal to tant traits in the latest monograph of the the mean length of the perfect-flowered in- (Hitchcock 1957)and are traits emphasizedin the dividuals. This eliminated segregation of in- latest regional flora (Hitchcock and Cronquist dividuals based solely on the sex of the flower. 1973).Sixteen morphological characterswere ini Data on the five parameters listed in Table I for tially examined (CH2M Hill 1986).Many of these S. nelsoniana, S. airgata, S. cusickii and S. characterswere judged to be so similar among campestris were analyzed by a PCA computer speciesas to be non-discriminatingand were not program (SAS Institute Inc. 1985).The raw data included in the investigation (e.g., calyx pu- from the specimensused in this analysisare sum- bescence,flower density, upper stem pubescence, marized in Table 2. style number). Other characters, while important taxonomically, are usually not found on most her- Following the PCA of the four species,a PCA barium specimens(e.g., root type, fruit traits). was performed on 73 specimens of Sidalcea nel- soniana alone, in order to examine the relation- Examination of the herbarium specimens ship between the specimens found in the Coast revealed that the flowers on the perfect-flowered Range, particularly those from Walker Flat, and plants tended to be larger than the flowers on those of the Willamette Valley. the pistillate-floweredplants. The influence of sex on the parameters used in the PCA was tested Resultsand Dascussion using a factorial analysis of variance (ANOVA) with flower sex and speciesas the factors. The The pollen of all specimensof Sidalcea is typical- number of specimensof each speciesand flower ly malvaceous (Figure l). The grains are 60-70 type are indicated in Table 2. This analysis in- microns in diameter, are conspicuously spiny, dicated a significant effect of flower sex and have a granular surface and are pantoporate.

Sidalcea Taxonomy 155 TABLE 2. Means and standard deviations of morphological parameters for Sidalcea species analyzed in the principal com- nonent analvsis.

Parameter'

Flower Species Sex n BASTPU PETCOL PETLG CALLC CALCOL

S. nelsoniana Perfect 4I 2.0+ 0.3 2.0r 0.0 I 1.5+ 2.0 5.5a 0.7 2.0r0.2 Pistillate 32 2.0+0.2 2.0a 0.0 7.1r 1.3 4.7+0.7 2.0+0.2

S. uirgata Perfect ll 3.0r 0.0 2.010.0 19.8r 3.6 8.7+ 1.4 1.7i 0.5 Pistillate l0 2.9r0.3 2.0+ 0.0 lu.6+ r.5 6.3i 1.2 1.8+ 0.4

S. cusickii Perfect 8 2.5t 0.9 1.9+ 0.4 I4.5i 3.5 7.8i2.0 1.9r 0.4 Pistillate 7 3.0t 0.0 2.0r 0.0 9.7+ 1.7 6.9r 0.8 1.8t 0.4

S. campestris Perfect l5 2.1!0.4 l.I r 0.2 17.3,t 4.6 7.5+1.3 1.7r0.5 Pistillate 7 2.0r 0.0 1.0+ 0.0 10.2t0.7 6.1r0.7 1.6+0.5

'See Table I for definitions of parameters.

The smooth,sticky surfacematerial coveringat dicates that the magnitude of the effect of flower leastpart of the granularsurface (Figure l-C,E) sex on petal and calyx length varies among is dueto thepresence of pollenkit(Dobson pers. species(Table 2). comm.),which has been reported from other The first two components of the PCA for the speciesof Sid,alcea(Dobson 1988). Although the four speciesof Sidalcea account for 70.6 percent pollenof S. campestrisexhibits some variability of the total variation, and are plotted in Figure in sizeand shapeof the spines,the pollengrains 2. The eigenvectorsfor each component are listed of all the taxa are very similar and do not ap- in Table 3. In general, there is an observable pear to be useful taxonomically. separation between S. nelsoniana in the left- The numberof chromosomesin all six of the central section of the plot, S. campestris in the S. nelsonianapopulations that were examined rightJower section, and S. uirgata in the right- wereidentical (n = l0). The specimenscounted upper of the figure. S. cusickii is less clearly wereHalse 3178, 3312 g amhill County,Walker segregated from the other species, and inter- Flat), Halse 33/3 (Tillamook County, Devils Lake mingles extensively with S. uirgata and somewhat Fork of the Wilson River), Halse 3289, 3293 with S. nelsoniana. These results suggest that, (Benton County, Corvallis),Halse 3240 (Polk with the exception of.S. cusickii, the characters County,Rickreall), Halse 3291 (Marion County, used in the PCA are generally effective and ap- Turner).This count is consistentwith an earlier propriate for distinguishing between the four report (Kruckeberg1957). S. nelsoniana,like S' speciesof Sidalcea used in this study. The third, cusickii,shows no signsof ,although fourth and fifth componentswere examined (plots polyploidyis found in both S. cam.pestris(2n = are not shown) and these also failed to provide 60) and S. rsirgata(2n = 20 or 40) (Kruckeberg a clearcut segregation between S. airgata and S. r9s7). cusickii. However, S. cusickii and S. airgatahave The results of the ANOVA indicate that there some distinguishing fruit and calyx characters (e.g., is a statistically significant difference (p < 0.001) not used in this study calyx venation and among speciesfor all of the five parameters listed shape, carpel reticulation). in Table l, indicating that all of them are useful One of the problems in identifying speciesof in distinguishing among species.The ANOVA Sidalcea from the Coast Range and Willamette also indicates statistically significant (p < 0.001) Valley is that the keys and species descriptions differences in petal length and calyx length be- in both Hitchcock's monograph (1957)and local tween the perfect-floweredand pistillate-flowered floras do not distinguish petal and calyx length plants of the same species.There is also a signifi- differences associated with flower sex. General- cant difference attributable to the species-by- ly the petal length, and calyx length, is simply flower sex interaction for the petal length (p ( given as the combination of the lengths of both 0.01) and the calyx length (p < 0.05). This in- sexes;i.e., in S. nelsoniana petal length is given

r56 Halse, Rottink, and Mishaga Figure l. Scanningelectron photomicrographs of Sidalceapollen. A, S. campestris,Polk Co.: Highway 22, Rickreall,Halse 3299;B, S. campestris,Linn Co.: Cogswell-Foster,Halse 3455; C, S. uirgata,Benton Co.: Smith Loop Road, south of Corvallis,Halse32Sl;D,S.cusickti,DouglasCo.:CalapooiaValley,Coles.n.;E,S. nelsoniana,YamhillCo.:Walker Flat, Halse 3312; F, Polk Co.: Rickreall, Halse 3290.(Voucher specimens at Oregon State University).

Sidalcea Taxonomy 157 PRINCIPALCOMPONENT 2 So o @s & a6r'Y 6g I R ! B € o F @ z a) (.) o !

n rf o EI 3 n 'ts oE E z fi L zm tr b D D D ;

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158 Halse, Rottink, and Mishaga TABLE 3. Eigenvectors for morphological parameters in each as 5-15 mm, rather than indicating that petal component of the principal component analysis length in perfect flowers is 9-15 mm, while petal of four species of Sidalcea. length in pistillate flowers is 5-9 mm. It is im- portant to account for flower sex, as both perfect- Component and pistillate-flowered plants are common. Ex- Parameter' amination of 32 populations of S. nelsoniana in- BASTPU +0.397 +0.542 0.259 -0.693 -0.032 dicated the average ratio of pistillate-flowered PETCOL 0.24r + 0.743 -0.277 +0.539 +0.148 plants to perfect-floweredplants is 1.36:l (CH2M -0.275 -0.185 CALCOL + 0.376 +0.864 0.043 Hill 1987). Incorrect identification can result ADPETLG + 0.599 + 0.000 +0.271 +0.208 +0.724 ADCALLG + 0.59I + 0.t14 +0.189 +0.388 -0.672 from using a key that fails to distinguish dif- ferencesin petal and calyx size associatedwith Variance flower sex. A key which allows for these dif- Explained 46.7% 23.9Vo 17.l% 93% 3.0% ferences, as well as utilizing other primary 'See Table I for definitions of oarameters characteristics,is presented below: l. Petalswhite to pink; basal stem pubescencesimple; petals of perfect flowers l3-25 mm long, calyx 6-9 mm long; petals of pistillateflowers 9-12 mm long, calyx 5-7 mm long...... 5. campestris l. Petals red, lavender or purple 2. Basal stem pubescencesimple; petals of perfect flowers 9-l5 mm long, calyx 4.5-7 mm long; petals of pistillateflowers 5-9 mm long, calyx 4-6 mm long...... S. nelsoniana 2. Basal stem pubescenceof forked to stellate hairs 3. Petals of perfect flowers ll-19 mm long, calyx 6-10 mm long; petals of pistillate flowers 8-12 mm long, calyx 6-8 mm long; calyx usually prominently veined, lobes widened above base, + ovate-lanceolate.... .5. cusicltii 3. Petals of perfect flowers l5-25 mm long, calyx 7-10 mm long; petals of pistillate flowers 9-13 mm long, calyx 5-7 mm long; calyx not prominently veined, lobes not widened above base, tapered evenly to the tip . . . . . S. oirgata

ThePCA using the five charactersexamined TABLE 4. Eigenvectors for morphological parameters in each on 73 specimensof S. nelsonianashows no com- component of the principal component analysis ponent separation among geographical popula- of Sidalcea nelsoniuna- tions of this species.The first two components Componenl of the PCA for S. nelsonia,?oare plotted in Figure Parameterr 3. The total variation in component I is similar in both Figure 2 and Figure 3. However, the BASTPU -0.205 +0.535 +0.807 +0.141 +0.000 overlap of the Willamette Valley and Coast PETCOL -0.000 +0.000 +0.000 +0.000 + 1.000 Range populations in Figure 3 along the com- CALCOL - 0.010 + 0.830 0.557 +0.026 +0.000 ponent I axis make it impossible to segregate ADPETLG +0.702 +0.016 +0.044 +0.710 +0.000 these groups. The first two componentsaccount ADCALLG +0.682 +0.157 +0.189 -0.689 +0.000 for 68.5 percent of the variation (Table 4). In Variance Figure 3, the specimensfrom the Coast Range Explained 43.t% 25.4% 24.2Vo 73% 0.0% and the Willamette Valley are intimately inter- 'See mixed, indicating that, based on the characters Table I for definitions of parameters. used in this PCA, the Willamette Valley and Coast Range populations of S. nelsoniana are not when the third and fourth components are ex- distinct morphologically. Similarly, the specimens amined (plots are not presented).These results from Walker Flat are closely intermixed with agree with electrophoretic analysis of both leaf other Coast Range specimens,indicating that the and seedproteins from Coast Range and Willam- population at Walker Flat is not a distinct taxon. ette Valley populations of S. nelsoniana (CH2M The Coast Range populations are also not differ- Hill 1986)which also show no differences among entiated from the Willamette Valley populations geographic populations of S. nelsoniana.

Sidalcea Taxonomy 159 - PRINCIPAI.COMPONENT 2

!" *l !5oN

d E=. €' ; o B & a! o ln ,a ;^\E= -t, s! 6 * !t @ in & F /^r o s l!, S- f- D F.> FO b 3-t 21 6m * :--5 ft-- { g 1-l J Y (1 "9 o

- =' € t

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160 Halse, Rottink, and Mishaqa An absolutely definitive statement on the Yalley Sidalcea. Recent concerns over the tax- genetic diversity between populations of S. nel- onomic status of these speciesmay have been in- soniana, and in particular between those at fluenced by oversight of the morphological varia- Walker Flat and other locations, is beyond the tion within gynodioecious species. The analysis scope of this study. However, to the extent that presented here also shows that S. nelsoniana is the taxonomic characteristicsstudied here are morphologically consistent throughout its range manifestationsof the plants' genetic makeup, this and so there is no basis for dividing the species study supports the hypothesis that the Walker into separate taxa. Flat population is not genetically distinct from other populations, and that the loss of the Walker Acknowledgments Flat population would not deplete the genetic We thank the City of McMinnville,Water and diversity of the species. Light Departmentfor financial support during In summary, analysisof pollen morphology thesestudies, which were contracted to CH2M and chromosomenumber, and the PCA of stan- Hill, Inc. We would alsolike to thank numerous dard morphological traits have been used to con- herbaria throughout the Pacific Northwestfor firm the speciesintegrity of the four Willamette permittingaccess to their collections.

Literature Cited Hitchcock,C. L. 1957.A study of the perennialspecies of Sidalcea.Univ. Wash. Pub. Biol. 18:l-79. CH2M Hill. 1985.Studies of Sidnlceanelsonana 1986.(Report Hitchcock,C. L., and A. Cronquist.1973. Flora of the Pacific submittedto U.S.Bureau of Land Management,Salem Northwest.University of WashingtonPress, Seattle. District and U.S. Fish and Wildlife Serviceby City Kruckeberg, A. R. 1957. Chromosomenumbers and in- of McMinnville, Departmentof Water and Light). terspecifichybridizations. Univ. Wash. Pub. Biol. 1987.Studies of Sidalceanelsoniano 1987. (Re- l8:83-93. port submittedto U.S. Bureauof Land Management, Roush,E. 1931.A monographof the gems Sidalcea.Ann. SalemDistrict and U.S. Fish and Wildlife Serviceby Mo. Bot. Gard. l8:ll7-224. City of McMinnville, Departmentof Water and Light). SAS Institute Inc. I985. SAS User's Guide: Statistics,Ver- Dobson,Heidi E. M. 1988.Survey of pollen and pollenkitt sion 5 Edition. Cary, N.C.: SAS Institute Inc. lipids-chemical cuesto flowervisitors? Amer. J. Bot. Snow,R. 1963.Alcoholic hydrochloricacid-carmine as a 75:170-1.82. stainfor chromosomesin squashpreparations. Stain Glad,Judith B., RichardMishaga, and RichardR. Halse.1987. Tech.38:9-13. Habitat Characteristicsof Sidalceanekoniana Piper U.S. Fish and Wildlife Service. 1985. Endangered and ()at Walker Flat, Yamhill County,Oregon. threatenedwildlife and plants;Review of plant taxa Northw. Sci. 6l:257-263. for listing as endangeredor threatenedspecies; Notice Halse.Richard R.. and Judith B. Glad. 1986.A note on the of Review.Federal Register50:39526-39584. statusof Sidalceanelsoniana (Malvaceae). Madrono Willson,M. F. 1983.Plant Reproductive Ecology. John Wiley 33:225-226. & Sons,Inc. New York.

Receioed 22 December 1987 Acceptedfor publication 1 February 1989

Sid.alceaTaxonomy 161