THE ERECTUM COMPLEX OF THE SOUTHERN APPALACHIANS

KATHY MATHEWS, PH.D.

PROFESSOR & HERBARIUM DIRECTOR

DEPARTMENT OF BIOLOGY

WESTERN CAROLINA UNIVERSITY

CULLOWHEE, NC 10 THINGS TO KNOW ABOUT

1. Trilliums are long-lived perennials that outcross 2. Trillium species are distinguished based on a variety of traits, including flower morphology, flowering time, and flower scent 3. Members of the Trillium erectum complex act like a single species – they can all interbreed & form intermediate, fertile offspring 1. TRILLIUMS ARE LONG-LIVED PERENNIALS THAT OUTCROSS

• A variety of insects visit flowers of Trillium, including butterflies, ants, yellowjackets, carpenter bees, bumblebees, sweat bees and flies • Insects sometimes eat the flower parts (petals, stigmas, styles and stamen) but leave the intact • There is a high degree of genetic variation within populations, indicating outcrossing (vs. selfing) • Fruits are berry-like capsules that fall off at maturity and break open to release the seeds • Seeds bear an elaiosome (“oil body”) which attract ants and other insects for dispersal (“myrmechochory”) • Seed dispersal is limited in distance Bear Pen Gap – 7/23/17. Photography by Ken Borgfeldt 2. TRILLIUM SPECIES ARE DISTINGUISHED BASED ON A VARIETY OF TRAITS, INCLUDING FLORAL MORPHOLOGY, FLOWERING TIME, AND FLORAL SCENT

TRILLIUM FLOWER SCENTS

• Wet dog, rotten meat (Trillium erectum) – “stinking Willie, stinking Benjamin” • Shoe polish/plastic shower curtain – T. tennesseenesis • Bananas (T. luteum) • Sweet, floral or fruity (T. cuneatum – “little Sweet Betsy,” T. simile – “sweet white Trillium,” T. vaseyi – “sweet Hamblen Co., TN Beth”) – SWEET WHITE TRILLIUM

• Flower upright, cup- shaped • Petals white, large and broad, slightly recurved at tips, concealing the stamens & ovary from side view • Ovary maroon • Scent: sweet, applelike

TRILLIUM ERECTUM – STINKING BENJAMIN• Wide-ranging species • Flowers are held above leaves • Clump-forming • Pedicels are long and straight • Petal colors are red or whitish • A crook in the pedicel causes flowers • Ovary is dark-colored to face forward (vs. upward) • Petals are relatively narrow • Flowers are “flat” like a plate (vs. cup- shaped) TRILLIUM ALBUM (T. ERECTUM VAR. ALBUM) GREAT SMOKY MOUNTAINS NATIONAL PARK, NEWFOUND GAP TRILLIUM ALBUM (T. ERECTUM VAR. ALBUM) – BALSAM MT. PRESERVE, SYLVA, NC

• Red variety is common throughout range • White variety is mainly found in southern Appalachians, typically in richer, less acidic soils and some high-elevation sites (Great Smokies)

SOUTHERN APPALACHIAN TRILLIUMS IN THE T. ERECTUM COMPLEX FLORAL CHARACTERISTICS TO HELP IDENTIFY “SPECIES”

1. Flower scent sweet or fetid 2. Flower upright or declined below leaves 3. Pedicel long or short, straight or curving 4. Flower flat like a plate or 3-dimensional like a cup 5. Petals white or maroon 6. Petals broad or narrow 7. Petals recurved from middle or below, or just at tips 8. Ovary light-colored or maroon 9. Ovary pyramidal or spherical 10. Filament/anther ratio: filament longer than anther, anther longer than filament, filament equal to anther TRILLIUM RUGELLII – SOUTHERN NODDING TRILLIUM

• Flowers declined below the leaves on a short, curved pedicel • Petals white, broad, recurved below middle • Ovary light-colored • Stamen filaments short (much shorter than anthers – SWEET BETH • Flowers and large! • Flowers declined below the leaves on a long pedicel • Petals and ovary dark maroon; Petals broad, recurved at tips • Stamen filaments long Photo by Hans Christensen.

Photo by Mark Pistrang, Cherokee National Forest. 3. MEMBERS OF THE TRILLIUM ERECTUM COMPLEX MAY BE REFERRED TO AS A SEMI-SPECIES OR SYNGAMEON • Defined by reproductive biologist, Verne Grant (1981), as having the following characteristics:

Definition of syngameon Characteristics of Trillium erectum complex No evidence of intrinsic reproductive They can all interbreed and form fertile barriers offspring Species-specific genetic markers have not been found Hybridization that could lead to Intermediate forms are commonly introgression found in areas of sympatry Evidence of mixed genotypes Distorted morphological boundaries It is often difficult to identify individuals to one species or another YOUR ETHNICITY ESTIMATE IS BASED ON YOUR OVERALL GENOTYPE AND TELLS YOU WHERE YOUR ANCESTORS MIGHT HAVE LIVED HUNDREDS OR THOUSANDS OF YEARS AGO.

Genotypes at one pair of chromosomes

DNA markers across all 23 pairs of chromosomes can identify groups of people who once lived in a geographic region for many generations without intermarrying with others from outside the area. FREDERICK W. CASE, JR. & ROBERTA B. CASE, TRILLIUMS, P. 97:

• “Because [T. erectum] intergrades with almost all related species, many specimens are impossible to key or place with confidence. That these confusing forms are hybrids we have verified by duplicating them with hand-pollinated crosses from selected typical plants of each species involved.” Example of a hybrid zone: Dark Cove Farm (Jackson County, NC): T. erectum, T. album, T. rugellii & T. vaseyi hybrids

HOW WE ARE STUDYING THE REPRODUCTIVE BIOLOGY AND ANCESTRAL HISTORY OF TRILLIUM ERECTUM

• Establishing parental genotypes: • Observe and collect data from single-species populations • Determine isolating mechanisms, if any: • Sample mixed populations with different flower colors. Do different flower colors attract different pollinators and affect interbreeding? • Sample near and far pure and mixed populations. Does geographic proximity affect interbreeding? POLLINATION STUDY (CHRISTINA STOEHREL 2010)

• Performed at Balsam Mountain Preserve • Pollen stained with histochemical dyes prior to anther dehiscence. • One white species • T. rugelii • Two red species • T. erectum • T. vaseyi (red T. rugelii?)

POLLINATION STUDY RESULTS

# of # of Color Species anthers anthers Pollinated by Pollinated by dyed collected white flower red flower

White T. rugelii 28 15 11 4

T. vaseyi 28 15 2 13 Red T. erectum 28 15 2 13

35.6% interspecific pollination Plant Red White Total pollen pollen 15.4% red pollinated by white Red 26 4 30

26.6% white pollinated by red White 4 11 15

X² = 3.84; df=1, p=0.05 Total 30 15 45

X² = 7.2; reject Hₒ that pollination was random GENETIC DATA COLLECTION

• Collect part of a leaf from up to 20 individuals per population • Collect one voucher specimen of each “type” to verify identity of sample • Grind up leaf tissue and extract DNA • Amplify variable genetic markers in the lab: microsatellites

SIX MICROSATELLITE LOCI DEVELOPED FOR JAPANESE RELATIVE, T. CAMSCHATCENSE

http://photos.sakhalin.name/photo/130083 Locus Repeat Motif Size (bp) Kubota, S, Kameyama, Y, Ohara, M. 2006. TC2 (GA)10T(AG)5(AC)10(AG)16 194-203 Characterization of six TC15 (GA) 235-279 microsatellite markers in Trillium 22 camschatcense using a dual- TC36 (AG)11(AC)8(AG)9AC(AG)8 191-205 suppression-polymerase chain reaction technique. Molecular TC44 (GA)8(CA)11 (GA)13 230-287 Ecology Notes 6:4 1135-1137 TC48 (TC) TT(TC) 148-182 15 4 TC69 (TC)10 . . .(AC)6(TC)9 167-187 Preliminary study 1 (small population samples, 1 marker single species populations)

CLDO (n=1) T. erectum var. album

BAMO (n=1) T. erectum var. album 304 100% T. erectum 310 293 50% 50% WOCR (n=3)

STIN (n=1) 304 289 33% 33%

308 280 298 293 50% 50% T. erectum 17% 17% 324 10% 301 WAWO (n=3) WHWA (n=5) 10% 280 20% 306 285 308 285 293 17% 16% 33% 33% 10% 285 20% BLRO (n=3) 301 289 289 17% 16% 30% 300 291 300 291 17% 17% 17% 17% T. vaseyi SAMPLING LOCALITIES FOR STUDY 2 – WE PLAN TO EXPAND TO SMOKIES, SOUTH OF Trillium CULLOWHEE & NORTHEAST OF ASHEVILLE

Untitled layer

WC-R Balsam Mountain Preserve Fleetwood Harmon Den Mountain BMP album BMP-R Fleetwood sampled pops for microsat Harmon Den

WCU BMP Preliminary study 2 (large population samples, mixed & pure sites) results: • Parental T. rugellii genotype (blue) found • Two parental T. erectum genotypes found: southern (red), northern (green) • T. erectum & T. album share same genotypes • Introgression from T. erectum into T. rugellii at BMP site.

Jackson County Ashe County

WCU rugellii BMP BMP BMP rugellii Fleetwood Harmon white red erectum red erectum Den white erectum erectum (album) BMP plants that look like ER & EA hybrids (album)

BMP=Balsam Mountain Preserve Future Work: • Gather data from more molecular markers to increase confidence in genotypes & ancestry analysis • Expand geographic range of population sampling • Include T. vaseyi & T. simile

Thank you for listening!