Research Article |
Corresponding author: Iwan E. Molgo ( imolgo@ufl.edu ) Academic editor: Luiz Gustavo Souza
© 2017 Iwan E. Molgo, Douglas E. Soltis, Pamela S. Soltis.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Molgo IE, Soltis DE, Soltis PS (2017) Cytogeography of Callisia section Cuthbertia (Commelinaceae). Comparative Cytogenetics 11(4): 553-577. https://doi.org/10.3897/compcytogen.v11i4.11984
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Determining the distribution of cytotypes across the geographic distribution of polyploid complexes can provide valuable information about the evolution of biodiversity. Here, the phytogeography of cytotypes in section Cuthbertia (Small, 1903) Hunt, 1986 is investigated. A total of 436 voucher specimens was georeferenced; 133 new specimens were collected. Based on flow cytometry data, DNA content of all cytotypes in section Cuthbertia was estimated. Utilizing chromosome counts and flow cytometric analysis, cytotype distribution maps were generated. Two disjunct groups of populations of diploid Callisia graminea (Small, 1903) Tucker, 1989 were discovered; tetraploid C. graminea ranges broadly from the coastal plain of North Carolina through central Florida. One hexaploid C. graminea individual was recorded in South Carolina, and numerous individuals of hexaploid C. graminea were found in central Florida. Diploid C. ornata (Small, 1933) Tucker, 1989 occurs in eastern Florida; previously unknown tetraploid and hexaploid populations of C. ornata were discovered in western and central Florida, respectively. Diploid C. rosea (Ventenat, 1800) Hunt, 1986 occurs in Georgia and the Carolinas, with populations occurring on both sides of the Fall Line. The cytotype and species distributions in Callisia are complex, and these results provide hypotheses, to be tested with morphological and molecular data, about the origins of the polyploid cytotypes.
chromosome counts, cytotypes, endemic, Florida scrub vegetation, flow cytometry, genome size, polyploidy, sandhill vegetation, Southeastern United States
Polyploidy (whole-genome duplication) is a speciation mechanism that is a major evolutionary force; in fact, all angiosperms have undergone at least one ancient polyploidy event (
Polyploids are classified in two major categories: allopolyploids and autopolyploids. Allopolyploids are by far the more studied form and arise via hybridization between species, whereas autopolyploids originate from the multiplication of genomes within a single species. An autopolyploid is frequently considered as a cytotype within a species along with its diploid progenitor, as in Galax urceolata (Poiret, 1804) Brummitt, 1972 (
To gain a better assessment of biodiversity and to guide conservation efforts for species of interest, data on both evolutionary and life-history characteristics are needed. Callisia section Cuthbertia (Commelinaceae) from the southeastern U.S.A. comprises a polyploid complex, with species of conservation concern, but the extent of polyploidy and the geographic distribution of cytotype diversity are unknown.
Callisia Loefling,1758 is one of 39 genera in subfamily Commelinoideae (
Callisia section Cuthbertia consists of three morphologically distinct species (C. graminea, C. ornata, and C. rosea) that are endemic to the southeastern U.S.A. and have a base chromosome number of x = 6 (
Although earlier studies (e.g.,
To obtain locality data for Callisia graminea, C. ornata, and C. rosea, voucher specimens were examined from the following herbaria: GA, USCH, NCU, DUKE, US, AAH,
Populations used in this study. Geographic location, ploidy, number of plants of each ploidy, total number of analyzed individuals, and voucher information for 133 populations of Callisia graminea (G), C. ornata (O), and C. rosea (R) from the southeastern United States. * indicates a new locality with voucher specimen.
Geographic coordinates | Ploidy / Number of plants | |||||||||
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Population | Locality | State | County | Latitude / Longitude | 2x | 4x | 6x | N | Voucher no. | |
Callisia graminea (Small) G. Tucker | ||||||||||
G-1* | Gainesville Regional Airport | FL | Alachua | 29°42.01'N, 082°15.72'W | 1 | 3 | 307 | |||
G-2 | Jct. Tower Rd. and SW 8 Ave | FL | Alachua | 29°38.63'N, 082°25.24'W | 1 | 4 | 223 | |||
G-3 | Morningside Nature Center | FL | Alachua | 29°39.56'N, 082°16.45'W | 1 | 1 | 234 | |||
G-4 | Jct. Hwy 200 and CR. 491 | FL | Citrus | 28°58.51'N, 082°21.84'W | 1 | 2 | 229 | |||
G-5* | Along Rod Rd. | FL | Clay | 30°01.52'N, 081°51.95'W | 1 | 1 | 225 | |||
G-6 | Golden Branch Head State Park | FL | Clay | 29°50.75'N, 081°57.04'W | 1 | 2 | 309 | |||
G-7 | Silver Sand Lake Rd. | FL | Clay | 29°47.49'N, 081°58.32'W | 1 | 4 | 311 | |||
G-8* | Tate Hell State Forest along New River | FL | Franklin | 29°52.42'N, 084°41.79'W | 1 | 4 | 306 | |||
G-9* | Richloam State Forest/Dark Stretch Rd. | FL | Hernando | 28°29.10'N, 082°08.87'W | 1 | 6 | 349 | |||
G-10* | Edwards Rd., Lady Lake | FL | Lake | 28°54.12'N, 081°53.40'W | 1 | 3 | 235 | |||
G-11* | Lake Griffin State Park | FL | Lake | 28°52.31'N, 081°53.41'W | 1 | 3 | 236 | |||
G-12* | Seminole State Forest along Co. Rd. 42 | FL | Lake | 29°00.82'N, 081°31.05'W | 1 | 1 | 3 | 345 | ||
G-13* | Seminole State Forest | FL | Lake | 28°49.31'N, 081°28.01'W | 1 | 1 | 362 | |||
G-14* | Lake Norris Rd. | FL | Lake | 28°54.89'N, 081°32.41'W | 1 | 1 | 363 | |||
G-15* | ATV trail at Ocala National Forest | FL | Marion | 29°21.76'N, 081°44.21'W | 1 | 1 | 230 | |||
G-16 | Silver River State Park | FL | Marion | 29°12.15'N, 082°02.77'W | 1 | 4 | 348 | |||
G-17* | Along Mason Rd. | FL | Putnam | 29°42.50'N, 082°00.77'W | 1 | 2 | 224 | |||
G-18* | Ordway Biological Center H1 & H2 area | FL | Putnam | 29°41.70'N, 081°57.87'W | 1 | 2 | 302 | |||
G-19* | Etoniah Creek State Forest | FL | Putnam | 29°46.43'N, 081°51.91'W | 1 | 3 | 308 | |||
G-20 | Dunns Creek State Park entrance Sisco Rd. | FL | Putnam | 29°31.84'N, 081°35.34'W | 1 | 4 | 310 | |||
G-21* | Welaka State Forest | FL | Putnam | 29°28.24'N, 081°39.37'W | 1 | 2 | 360a | |||
G-22 | Along State Rd. 46 | GA | Bulloch | 32°20.94'N, 081°50.57'W | 1 | 3 | 242 | |||
G-23 | Jct. Hwy 185 and Turkey Ridge Dr. | GA | Charlton | 30°24.76'N, 082°11.70'W | 1 | 2 | 317 | |||
G-24* | General Coffee State Park | GA | Coffee | 31°31.50'N, 082°46.33'W | 1 | 1 | 318 | |||
G-25 | N. Connector Rd./206 Jct. 135 | GA | Coffee | 31°32.27'N, 082°46.33'W | 1 | 3 | 319 | |||
G-26* | George Smith State Park | GA | Emanuel | 32°32.64'N, 082°07.32'W | 1 | 6 | 241 | |||
G-27* | Ochicoo Preserve, Halls Bridge Rd. | GA | Emanuel | 32°31.73'N, 082°27.38'W | 1 | 4 | 320 | |||
G-28 | Fort Stewart | GA | Evans | 32°06.92'N, 081°47.10'W | 1 | 4 | 243 | |||
G-29* | Conway CT./Interstate Parkway | GA | Richmond | 33°29.24'N, 082°06.12'W | 1 | 1 | 322 | |||
G-30 | Fort Gordon | GA | Richmond | 33°23.33'N, 082°14.56'W | 239 | |||||
G-31* | Singletary Lake State Park | NC | Bladen | 34°35.41'N, 078°26.87'W | 1 | 3 | 263 | |||
G-32* | Jones Lake State Park | NC | Bladen | 34°42.11'N, 078°37.22'W | 1 | 3 | 268 | |||
G-33* | Jones Lake State Park | NC | Bladen | 34°42.11'N, 078°37.22'W | 1 | 269 | ||||
G-34* | Along NC 242 near Jones Lake State Park | NC | Bladen | 34°42.00'N, 078°36.35'W | 1 | 2 | 270 | |||
G-35* | Along NC 242 N. of Jones Lake State Park | NC | Bladen | 34°45.40'N, 078°36.56'W | 1 | 5 | 271 | |||
G-36* | White Lake, along NC 741, Barnes Food Co. | NC | Bladen | 34°39.41'N, 078°30.17'W | 1 | 5 | 272 | |||
G-37* | Jones Lake State Park. campsite | NC | Bladen | 34°40.79'N, 078°35.99'W | 274 | |||||
G-38* | Along Burney Rd. underneath powerline | NC | Bladen | 34°44.38'N, 078°43.68'W | 1 | 4 | 334 | |||
G-39* | River Rd., underneath powerline | NC | Bladen | 34°46.18'N, 078°47.24'W | 1 | 3 | 335 | |||
G-40 | Bay Tree Lake State Park/undeveloped | NC | Bladen | 34°40.22'N, 078°25.66'W | 1 | 6 | 261 | |||
G-41 | Along Hwy 41 close to Bay Tree Lake State Park | NC | Bladen | 34°41.21'N, 078°25.26'W | 1 | 3 | 262 | |||
G-42 | Along Hwy 11 towards Delco under powerline | NC | Bladen | 34°24.61'N, 078°15.60'W | 1 | 4 | 266 | |||
G-43 | Along Jessup Pond | NC | Bladen | 34°51.72'N, 078°43.76'W | 275 | |||||
G-44 | Lake Waccamaw State Park. | NC | Columbus | 34°16.73'N, 078°27.89'W | 267 | |||||
G-45* | Mack Simmons Rd. | NC | Cumberland | 34°54.45'N, 078°44.20'W | 276 | |||||
G-46* | Along NC 210, Jct. with Sidney Bullard Rd. | NC | Cumberland | 34°58.69'N, 078°43.84'W | 1 | 4 | 278 | |||
G-47* | Ft. Bragg/John Mill Rd. | NC | Cumberland | 35°10.70'N, 079°05.39'W | 1 | 3 | 341 | |||
G-48* | Ft. Bragg/NE. training/Mc Closkey Rd. | NC | Cumberland | 35°09.84'N, 078°56.97'W | 1 | 3 | 342 | |||
G-49 | Cedar Creek Rd., Tatum farm | NC | Cumberland | 34°56.32'N, 078°44.58'W | 1 | 1 | 277 | |||
G-50 | Along Dunns Rd./NC 301 | NC | Cumberland | 35°06.42'N, 078°46.52'W | 279 | |||||
G-51 | Open Area along NC 24 | NC | Harnett | 35°15.61'N, 079°02.47'W | 1 | 3 | 284 | |||
G-52 | Along Rockfish Rd. | NC | Hoke | 34°59.32'N, 079°05.82'W | 1 | 3 | 286 | |||
G-53 | In open area along Red Springs Rd. | NC | Hoke | 34°52.38'N, 079°12.17'W | 1 | 4 | 287 | |||
G-54* | Weymouth Sandhill Nature Preserve | NC | Moore | 35°08.95'N, 079°22.10'W | 1 | 3 | 288 | |||
G-55 | Along Riverview Dr. | NC | Moore | 35°11.48'N, 079°10.94'W | 1 | 3 | 285 | |||
G-56 | Along NC 11/ Hwy 53 | NC | Pender | 34°29.72'N, 078°11.49'W | 1 | 3 | 264 | |||
G-57 | Along NC 11/ Hwy 53 | NC | Pender | 34°29.72'N, 078°11.49'W | 1 | 1 | 265 | |||
G-58* | Grey Woods Rd. | NC | Richmond | 34°57.52'N, 079°38.47'W | 1 | 3 | 297 | |||
G-59* | Sandhills Game Land | NC | Richmond | 35°01.83'N, 079°36.70'W | 1 | 2 | 336 | |||
G-60* | Sandhills Game Land/442/Ledbetter Rd. | NC | Richmond | 35°03.62'N, 079°38.09'W | 1 | 3 | 337 | |||
G-61* | Sandhills Game Land | NC | Richmond | 34°58.61'N, 079°30.42'W | 1 | 2 | 338 | |||
G-62* | Sandhills Game Land SR 1331, 15/501 | NC | Richmond | 34°58.50'N, 079°26.93'W | 1 | 2 | 339 | |||
G-63* | Sandhills Game Land, Aberdeen Rd./Hill Creek Rd. | NC | Richmond | 34°59.49'N, 079°26.76'W | 1 | 3 | 340 | |||
G-64 | Sandhills Game Land along McDonald Church Rd. | NC | Richmond | 35°01.24'N, 079°37.18'W | 1 | 2 | 290 | |||
G-65 | NC Hwy 177 | NC | Richmond | 34°50.41'N, 079°45.54'W | 1 | 1 | 295 | |||
G-66 | Along Saint Stevens Church Rd. | NC | Richmond | 34°49.82'N, 079°50.55'W | 1 | 1 | 296 | |||
G-67 | NC 242, 0.3 mi N. of Cumberland Co. line | NC | Sampson | 34°53.35'N, 078°31.28'W | 1 | 3 | 273 | |||
G-68 | Along Spiveys Corner Hwy. | NC | Sampson | 35°10.72'N, 078°28.65'W | 1 | 2 | 280 | |||
G-69 | Edge camp Mackall along Aberdeen Rd. | NC | Scotland | 35°00.84'N, 079°26.70'W | 1 | 2 | 289 | |||
G-70 | Along 1328, Hoffman Rd./Butler Rd. | NC | Scotland | 34°59.14'N, 079°31.99'W | 1 | 2 | 291 | |||
G-71 | Along Peach Orchard Rd. under powerline | NC | Scotland | 34°55.77'N, 079°23.86'W | 1 | 3 | 292 | |||
G-72 | Along US 401 and forest edge | NC | Scotland | 34°50.49'N, 079°23.98'W | 1 | 1 | 293 | |||
G-73 | Along forest edge of Hamlet Rd. | NC | Scotland | 34°48.01'N, 079°38.03'W | 1 | 2 | 294 | |||
G-74 | Along Piney Grove Church Rd. | NC | Wayne | 35°17.32'N, 077°50.92'W | 1 | 1 | 281 | |||
G-75* | Aiken State Park | SC | Aiken | 33°32.55'N, 081°28.92'W |
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1 | 4 | 324 | ||
G-76* | Parcel at Jct. Hwy 283 & US 1/Columbia Hwy N | SC | Aiken | 33°36.11'N, 081°41.04'W | 1 | 5 | 325 | |||
G-77 | Aiken Gopher Tortoise Heritage Preserve | SC | Aiken | 33°30.00'N, 081°24.52'W | 1 | 1 | 231 | |||
G-78* | Carolina Sandhills National Wildlife Refuge | SC | Chesterfield | 34°31.46'N, 080°13.63'W | 1 | 3 | 331 | |||
G-79* | Sandhill State Forest | SC | Chesterfield | 34°33.37'N, 080°03.84'W | 1 | 3 | 332 | |||
G-80* | H. Cooperblack Jr. Memorial trail/James Rd. | SC | Chesterfield | 34°34.03'N, 079°55.75'W | 1 | 2 | 333 | |||
G-81 | Along Hwy 102 | SC | Chesterfield | 34°38.30'N, 080°05.22'W | 1 | 5 | 249 | |||
G-82 | Teals mill Rd./Cheraw State Park | SC | Chesterfield | 34°37.25'N, 079°56.70'W | 1 | 1 | 3 | 250 | ||
G-83 | W. Old Camden Rd. | SC | Chesterfield | 34°22.28'N, 080°16.92'W | 1 | 3 | 252 | |||
G-84 | US 1 | SC | Chesterfield | 34°26.17'N, 080°17.44'W | 1 | 2 | 253 | |||
G-85 | Along Old Stagecoach Rd. | SC | Chesterfield | 34°20.96'N, 080°21.27'W | 1 | 3 | 254 | |||
G-86 | Along Old Georgetown Rd. E. | SC | Chesterfield | 34°22.99'N, 080°23.29'W | 1 | 1 | 255 | |||
G-87 | Co. Rd. S. 18-137 | SC | Dorchester | 32°54.02'N, 080°23.11'W | 1 | 4 | 248 | |||
G-88 | Tillman Sand Ridge Heritage Preserve, Sandhill Rd. | SC | Jasper | 32°29.69'N, 081°11.55'W | 1 | 5 | 247 | |||
G-89* | Along Jefferson Davis Hwy/US 1 | SC | Kershaw | 34°18.73'N, 080°32.49'W | 256 | |||||
G-90* | Goodale State Park | SC | Kershaw | 34°17.42'N, 080°31.55'W | 1 | 3 | 329 | |||
G-91* | Jefferson Davis Hwy/US 1 | SC | Kershaw | 34°22.04'N, 080°25.92'W | 1 | 4 | 330 | |||
G-92* | Lee State Park | SC | Lee | 34°11.81'N, 080°11.36'W | 1 | 3 | 251 | |||
G-93 | Shealy’s Pond Heritage Preserve | SC | Lexington | 33°51.82'N, 081°14.19'W | 1 | 1 | 232 | |||
G-94 | Peachtree Rock Preserve | SC | Lexington | 33°49.71'N, 081°12.11'W | 1 | 1 | 233 | |||
G-95* | Ft. Jackson, Area 26 B firebreak 16 | SC | Richland | 34°00.85'N, 080°47.40'W | 1 | 2 | 257 | |||
G-96* | Ft. Jackson, Area 34 B near Chauers Pond Rd. | SC | Richland | 34°02.36'N, 080°43.30'W | 1 | 3 | 258 | |||
G-97* | Ft. Jackson, Area 11 E. of Wildcat Rd. | SC | Richland | 34°05.06'N, 080°50.61'W | 1 | 2 | 259 | |||
G-98 | Ft. Jackson, S. edge of pond of Westons Recreation | SC | Richland | 33°59.96'N, 080°50.03'W | 1 | 1 | 260 | |||
G-99 | Sesquicentennial State Park | SC | Richland | 34°05.82'N, 080°54.57'W | 1 | 3 | 326 | |||
G-100* | Sesquicentennial State Park | SC | Richland | 34°04.92'N, 080°54.38'W | 1 | 1 | 4 | 327 | ||
G-101 | Faunas Rd. | SC | Richland | 34°08.34'N, 081°02.33'W | 1 | 5 | 328 | |||
G-102* | Forks of River Rd. | VA | Southampton | 36°33.85'N, 076°55.96'W | 1 | 2 | 282 | |||
G-103 | Suffolk City, DCR | VA | Suffolk City | 36°33.77'N, 076°54.82'W | 283 | |||||
Callisia ornata (Small) G. Tucker | ||||||||||
O-1* | Turkey Creek Sanctuary | FL | Brevard | 28°01.01'N, 080°36.18'W | 1 | 1 | 315 | |||
O-2* | Sebastian State Park | FL | Brevard | 27°50.19'N, 080°31.56'W | 1 | 2 | 361 | |||
O-3 | Wickham Park | FL | Brevard | 28°09.64'N, 080°39.54'W | 1 | 1 | 314 | |||
O-4* | Highlands State Park | FL | Highlands | 27°28.85'N, 081°31.57'W | 1 | 4 | 301 | |||
O-5 | Sebring Amtrak Station | FL | Highlands | 27°29.75'N, 081°26.06'W | 298 | |||||
O-6 | Lake June in Winter Scrub State Park | FL | Highlands | 27°17.83'N, 081°25.14'W | 1 | 2 | 300 | |||
O-7 | Little Manatee State Park/Mustang trail | FL | Hillsborough | 27°40.08'N, 082°22.1'W | 1 | 4 | 350 | |||
O-8 | Little Manatee State Park/Dude trail | FL | Hillsborough | 27°39.93'N, 082°22.38'W | 1 | 3 | 351 | |||
O-9* | Seminole State Forest/entrance Brantley Branch Rd. | FL | Lake | 28°53.20'N, 081°27.60'W | 1 | 4 | 343 | |||
O-10* | Seminole State Forest/the Simson track | FL | Lake | 28°52.94'N, 081°31.08'W | 1 | 4 | 344 | |||
O-11* | Seminole State Forest/Warea tract | FL | Lake | 28°29.99'N, 081°40.03'W | 1 | 3 | 346 | |||
O-12* | Lake Louisa State Park/Primitive campsite | FL | Lake | 28°27.17'N, 081°44.13'W | 1 | 4 | 347 | |||
O-13 | Jonathan Dickinson State Park/Nature trail picnic area | FL | Martin | 26°59.58'N, 080°08.83'W | 1 | 4 | 353 | |||
O-14* | Tiger Creek Preserve along Pfundstein Rd. | FL | Polk | 27°48.41'N, 081°29.81'W | 1 | 1 | 228 | |||
O-15* | Arbuckle State Forest, School Bus Rd. | FL | Polk | 27°39.75'N, 081°23.84'W | 1 | 3 | 316 | |||
O-16* | Lake Kissimmee State Park, Buster Island | FL | Polk | 27°55.39'N, 081°21.82'W | 1 | 2 | 354 | |||
O-17* | Lake Kissimmee State Park, Catfish Creek | FL | Polk | 27°57.84'N, 081°22.77'W | 1 | 5 | 355 | |||
O-18* | Lake Kissimmee State Park Main entrance | FL | Polk | 27°57.91'N, 081°28.34'W | 1 | 5 | 356 | |||
O-19* | Welaka State Forest | FL | Putnam | 29°28.24'N, 081°39.37'W | 1 | 1 | 360B | |||
O-20 | Dunns Creek State Park entrance Sisco Rd. | FL | Putnam | 29°33.34'N, 081°34.94'W | 1 | 2 | 312 | |||
O-21 | Oscar Scherer State Park along Legacy trail | FL | Sarasota | 27°10.17'N, 082°27.41'W | 1 | 5 | 352 | |||
O-22* | Tiger Bay State Forest | FL | Volusia | 29°10.22'N, 081°09.56'W | 1 | 3 | 313 | |||
O-23* | Lake George State Forest | FL | Volusia | 29°11.84'N, 081°30.55'W | 1 | 1 | 364 | |||
O-24* | Deland | FL | Volusia | 29°00.11'N, 081°13.25'W | 1 | 1 | 365 | |||
Callisia rosea (Vent.) D.R. Hunt | ||||||||||
R-1 | Along Chert Quarry Rd. | SC | Allendale | 33°02.28'N, 081°28.26'W | 1 | 3 | 245 | |||
R-2* | Heggie’s Rock Preserve | GA | Colombia | 33°32.34'N, 082°15.09'W | 1 | 3 | 321 | |||
R-3* | Lake Russel State Park | GA | Elbert | 34°09.60'N, 082°44.42'W | 1 | 3 | 237 | |||
R-4* | Bobbie Brown State Park | GA | Elbert | 33°58.35'N, 082°34.64'W | 1 | 3 | 238 | |||
R-5* | Elijah Clarke State Park | GA | Lincoln | 33°51.22'N, 082°24.02'W | 1 | 3 | 323 | |||
R-6 | Fort Gordon | GA | Richmond | 33°23.49'N, 082°14.54'W | 1 | 3 | 240 | |||
R-7 | Fort Stewart | GA | Tattnall | 32°02.54'N, 081°48.84'W | 1 | 4 | 244 |
The georeferenced data were used to relocate populations within the southeastern U.S.A.; additional localities were discovered by exploring similar habitats in protected areas and on private land. Collections on private land were made with permission of the land owners. Based on the georeferenced data, permits were obtained to collect in state parks, state forests, national parks, and protected areas of The Nature Conservancy and the U.S. Fish and Wildlife Service in Florida, Georgia, South Carolina, North Carolina, and Virginia (Table
Mature individuals were sampled in the summers of 2012, 2013, 2014, and 2015. Only known localities with collection years between 1970 and 2012 were visited, unless the locality was in a protected area. This approach was used to increase the chances of finding intact populations but meant that we were unable to resample all of
Population localities were surveyed for individuals with different growth habit and habitat; we then collected across that diversity. Contact zones between species, based on the georeferenced localities, were more intensively surveyed by searching for distinct morphological variation (habit, leaf, and flower) to increase the probability of encountering mixed cytotypes. Two to six live plants were collected per locality. Plants were removed with 15 cm of soil circumference to increase the survival rate and placed in plastic bags. At the Department of Biology, University of Florida greenhouse, plants were then potted in a soil mixture of 1:1 sand and potting soil (Pro-Mix) and were kept under natural light. During the period from December–March, the individuals of putative diploid C. graminea and C. rosea were given a four-month dormancy treatment at 4°C to mimic their natural habitat.
Two individuals per cytotype of C. graminea were used as a control for flow cytometry analysis by counting chromosome numbers using established methods (see below). Previous studies of members of Commelinaceae found that cell division in root tips occurs at high frequency during late morning to early afternoon (
Root tips were placed in 2 mM 8-hydroxyquinoline following
Preparation of all samples for flow cytometry followed
For the estimation of genome size, three plants of the same accession were analyzed using the Flow Cytometry Kaluza Analysis Software 1.3 (Beckman Coulter Life Sciences 2016). The relative DNA content was calculated using the ratio of the mean fluorescent peak of the sample to the mean fluorescent peak of the internal standard, multiplied by the genome size of the standard, Vicia faba (
All GPS points obtained here were incorporated into a map with ARCGIS 10.4 (
Distribution map of Callisia section Cuthbertia. Distribution of Callisia graminea, C. ornata, and C. rosea based on georeferenced data. Multiple species occurring in sympatry are designated by superimposed symbols; these locations are further indicated by black lines that highlight the symbols.
Chromosome numbers were obtained for three individuals per cytotype in C. graminea, confirming the presence of 2n = 2x = 12 (diploids; Figure
Mitotic metaphase chromosome spreads from root tips. A diploid Callisia graminea (2n = 2x = 12) B tetraploid C. graminea (2n = 4x = 24) C hexaploid C. graminea (2n = 6x = 36) D diploid C. ornata (2n = 2x = 12) E tetraploid C. ornata (2n = 4x = 24) F hexaploid C. ornata (2n = 6x = 36) and G diploid C. rosea (2n = 2x = 12).
Ploidy was estimated via flow cytometry for 300 plants of C. graminea (representing 96 populations), C. ornata (from 23 populations), and C. rosea (from 7 populations). The results and the number of individuals analyzed per population are given in Table 1. Three distinct groups of fluorescence intensities were obtained from these analyses that were congruent with chromosome counts of diploid, tetraploid, and hexaploid C. graminea. Histograms for the cytotypes of C. graminea are shown in Figure
Histograms of fluorescence intensity (FL2-A) of propidium iodide-stained nuclei. A diploid C. graminea B tetraploid C. graminea and C hexaploid C. graminea. Vicia faba was used as the internal standard.
The relative genome size of individuals of C. rosea was similar to that of diploid C. graminea (2n = 2x = 12) (see below), confirming that our samples of C. rosea are diploid, in agreement with the literature (
Genome size (2C-value) of cytotypes in Callisia section Cuthbertia was estimated; data are presented in Table
Genome sizes (2C) of Callisia section Cuthbertia and their cytotypes and previously reported 2C-values. Voucher numbers apply only to the current study.
Species | Chromosomes | 2C value (pg) |
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|
---|---|---|---|---|
C. graminea 2x (IEM 342) | 2n = 12 | 41.75 ± 0.67 | ||
C. graminea 4x (IEM 251) | 2n = 24 | 78.55 ± 0.42 | ||
C. graminea 6x (IEM 236) | 2n = 36 | 122.86 ± 0.8 | ||
C. ornata 2x (IEM 353) | 2n = 12 | 48.51 ± 1.09 | ||
C. ornata 4x (IEM 352) | 2n = 24 | 87.99 ± 0.4 | ||
C. ornata 6x (IEM 349) | 2n = 36 | 129.73 ± 0.56 | ||
C. rosea 2x (IEM 237) | 2n = 12 | 43.70 ± 1.78 | 43.52 | 77.3 |
Distribution map – Based on the flow cytometry data, the distribution of cytotypic variation among the 126 populations sampled [C. graminea (96 populations), C. ornata (23 populations), and C. rosea (7 populations)] was mapped (Figure
Distribution of cytotypic variation in C. allisia section Cuthbertia. Diploid C. graminea (red circles) ranges from Virginia to North and South Carolina; tetraploid C. graminea (purple circles) occurs along the coastal plain from North Carolina to central Florida; hexaploid C. graminea (black plus signs) is restricted to central Florida. Diploid C. ornata (red squares) occurs in eastern and central Florida; tetraploid C. ornata (purple squares) is restricted to central and western peninsular Florida; hexaploid C. ornata (green plus signs) is restricted to central Florida. Callisia rosea (all diploid; green diamonds) occurs along the Georgia – South Carolina border. Localities with multiple cytotypes or taxa are indicated by black lines. Note: The black plus signs are the hexaploids of C. graminea, and the green plus signs are hexaploids of C. ornata
Diploid C. ornata occurs in eastern Florida (from Putnam through Martin Counties), and tetraploid C. ornata occurs in western Florida (Polk, Hillsborough, Highlands, and Lake Counties). Hexaploid C. ornata occurs in Lake and Volusia Counties in central Florida.
Diploid C. rosea occurs in the piedmont of Georgia and South Carolina with some scattered populations in the coastal plain.
Georeferencing – Callisia section Cuthbertia consists of three species native to the southeastern U.S.A., with three ploidal levels within C. graminea and C. ornata and diploids in C. rosea. The map of the geographic distribution (Figure
Flow cytometry and genome size – Flow cytometry analysis of ploidal levels in 300 individuals from 126 populations together with 60 additional chromosome counts confirmed the presence of diploid, tetraploid, and hexaploid cytotypes of C. graminea and C. ornata. Significantly, tetraploid and hexaploid C. ornata were previously unknown. Our analysis also confirmed that C. rosea is diploid. However,
It is interesting to note that 26 individuals (17%) of tetraploid C. graminea had a lower fluorescence intensity than the remaining 83%, suggesting a smaller genome size. The individuals with the smaller peak than that typical of other tetraploids were measured twice with the flow cytometer, and the results were consistent. The chromosome numbers of these samples were verified by chromosome counts, and all were tetraploid (2n = 4x = 24). Reductions in genome size in polyploids are common (
Genome size can be used, with other methods, to hypothesize putative progenitors of polyploids (e.g.
Due to the rarity of hexaploid C. graminea in South Carolina, we only calculated the 2C-value of hexaploids that occur in Florida. Hexaploid C. graminea may be of allo- or autopolyploid origin. If from allopolyploid origin, the expected 2C-value would be 127.06 pg, with diploid C. ornata (48.51 pg) and tetraploid C. graminea (78.55 pg) as the progenitors. The observed genome size of hexaploid C. graminea is 122.86 pg, which is lower than the expected value, again consistent with genome downsizing. In the case of an autopolyploid origin with tetraploid C. graminea (78.55 pg) as parent, we would expect a genome size of 117.83 pg, which is approximately 5 pg less than the observed 2C-value. Genome size data do not conclusively elucidate the origins of hexaploid C. graminea; both allo- and autopolyploidy are possible, and its origin requires further testing. However,
Tetraploid C. ornata has a 2C-value of 87.99 pg. It could be of autopolyploid origin with diploid C. ornata (48.51 pg) as the parent given that no other extant taxa are sympatric with it. However, the expected DNA content (97.02 pg) is at least 9 pg higher than observed; in contrast, when considering tetraploid C. ornata as a possible allopolyploid with tetraploid C. graminea (78.55 pg) and diploid C. ornata (48.51 pg) as parents (based on an unreduced gamete of the latter), the results (87.79 pg) are similar to the observed DNA content. These results therefore support allopolyploidy over autopolyploidy, yet further analyses are needed to clarify the origin of this cytotype.
Hexaploid C. ornata could be of allo- or autopolyploid origin. If allopolyploid, the expected genome size would be 127.06 pg with diploid C. ornata (48.51 pg) and tetraploid C. graminea (78.55 pg) as parents. The observed DNA content is 129.73 pg, which is slightly higher than the expected 2C-value. Alternatively, it could be an allohexaploid between tetraploid C. ornata (87.99 pg) and diploid C. graminea (41.75 pg), with an expected genome size of 129.74 pg, essentially identical to the observed value. In the case of autopolyploidy, we calculated an expected 2C-value of 145.53 if the value is 3 times that of diploid C. ornata (48.51 pg), 136.5 pg if tetraploid (87.99 pg) and diploid (48.51 pg) C. ornata are considered the parents, and 131.99 pg if a reduced and unreduced gamete of tetraploid C. ornata yield the hexaploid. The latter case is closest to the observed value, suggesting either that hexaploid C. ornata is of allopolyploid origin, or if an autopolyploid, it arose via the third possible mechanism outlined above; these hypotheses require further investigation.
Based on the Plant DNA C-values Database, http://data.kew.org/cvalues/ (
Distribution – As shown in Figure
Tetraploid C. graminea ranges from the coastal plain of the Carolinas to central Florida, with additional populations in the Florida panhandle (Franklin County, FL). This cytotype is clearly more abundant than diploid C. graminea; it is usually found in xeric disturbed areas and exhibits a larger growth form than diploid C. graminea. These tetraploids were abundant in Bladen and southern Cumberland Counties, NC, which border the isolated locality of diploid C. graminea in North Carolina. These two areas (occupied by tetraploid and diploid plants, respectively) are separated by the city of Fayetteville, NC. Although diploid and tetraploid entities of C. graminea were reported to be geographically isolated (
The Fall Line runs essentially east-west through Georgia and from southwest to northeast in the Carolinas. Diploid C. rosea occurs on both sides of the Fall Line from Georgia to North Carolina. In Fort Gordon (Richmond County, GA), diploid C. rosea occurs in sympatry with tetraploid C. graminea. Although these two species occur in sympatry, hybrids were not observed at the site.
Diploid C. ornata is endemic to Florida, and tetraploid individuals of C. ornata occur in western Florida. These individuals may be autopolyploid, with diploid C. ornata as their progenitor. The distribution map in Figure
In South Carolina, one hexaploid individual of C. graminea was found growing sympatrically with multiple tetraploid individuals of C. graminea. Hexaploid C. graminea in South Carolina appeared to be rare, and in 1942 only one individual was reported by
In Lake and Hernando Counties, FL, hexaploid individuals exhibited intermediate morphological characteristics between C. graminea and C. ornata. Some populations had typical tetraploid C. graminea or diploid C. ornata characteristics (Figure
The rare hexaploid collected in South Carolina is most likely independently evolved from the hexaploids from Florida, and this entity from South Carolina could be either an allo- or autopolyploid. If allopolyploid, one likely parent, C. rosea, only occurs in Georgia and the Carolinas; if autopolyploid, the likely parent is tetraploid C. graminea. The hexaploid entities of Florida might be allopolyploid due to the intermediate morphological characters, with diploid C. ornata and tetraploid C. graminea as progenitors.
Callisia graminea forma leucantha, which was reported near Tampa, FL, was not found, but one white-flowered tetraploid individual of C. graminea was encountered among pink-flowered individuals in each of the following three locations: Sesquicentennial State Park, SC; Chesterfield Co., SC; and Tate’s Hell State Forest, FL. One white-flowered individual of diploid C. rosea was found in Heggie’s Rock Preserve, Appling, GA. White flowers reflect an absence of anthocyanins, which may result from mutations in any of the genes in the anthocyanin pathway or from lack of expression of potentially functional genes (
Morphological and molecular analysis is an important next step in unraveling the complex relationships among cytotypes of Callisia section Cuthbertia. This work will allow us to reveal the parentage, evolutionary history, and the evolutionary role of all cytotypes within Callisia section Cuthbertia.
The authors thank the curators of the following herbaria for access to the information on the voucher specimens of Callisia section Cuthbertia: GA, USCH, NCU, DUKE, US, AAH,
Georeferenced data points
Data type: occurence