Dirca

Abstract: The genus Dirca consists of three disjunct species of shrubs. Dirca palustris is found in the eastern United States and adjacent Canada; D, occidcntahs is Umited to six counties near the San Francisco Bay in California; and the recently discovered D, mcxicana is known from one isolated population in northeastern Mexico. The three species have been described and classified according to morphological characters, but the morphological evidence does not provide a clear assessment of the relationships among the species. Morphologically D. mexicana most closely resembles D. occidenlalis., but known biogeographical trends raise doubt regarding how the three species are interrelated. We used molecular techniques to examine and clarify phylogenetic relationships among the three species of Dirca. Evidence from Internal Transcribed Spacer (ITS) sequences and Inter-Simple Sequence Repeats (ISSR) polymorphisms confirms the species-level divergence of Dirca mexicana and reveals that, despite their morphological similarity D. mexicana and D. occidenlalis -cue the most divergent of the three species genetically while D, me.xicana and D. palustris are the most closely related. The molecular evidence also demonstrates that Dirca occidenlalis was the first of the three species to diverge and has undergone the greatest degree of differentiation since divergence. RESUMEN El genero Diixa consta de tres especies de arbustos separadas geograficamente. Dirca palustris se encuentra en la parte Este de los Estados Unidos y la parte contigua de Canada; D. occidcntalis se limita a seis condados alrededor de la bahia de San Francisco en California, EE.UU.; y la recien descubierta D. mexicana se conoce solamente de una poblacion aislada en la parte noreste de Mexico. Las tres especies han sido descritas y clasificadas segun sus caracteristicas morfologicas, pero las pruebas morfologicas han sido consideradas como no concluyentes para hacer una evaluacion clara de las relaciones entre las especies. Morfologicamente, D. mexicana se parece mas a D. occidenlalis, pero las tendencias biogeograficas establecidas crean dudas sobre el grado de parentesco entre las dos especies. Hemos usado tecnicas moleculares para examinar y clarificar las relaciones I ilogeneticas entre las tres especies de Dirca. Por las pruebas que encontramos en las secuencias del Espaciador Transcrito Interne (ETI) [Internal Transcribed Spacer (ITS)| y polimorfismos de Repeticiones de Secuencias Intra-Simple (RSIS) [Inter-Simple Sequence Repeats (ISSR)], Uegamos a las siguientes conclusiones; 1. Dirca mexicana merece ser catalogada como especie; 2. Dirca occidenlalis fue la primera de las especies en evolucionar, y ha experimentado el mayor grado de diierenciacion desde su divergencia; y 3. Contrario a sus morfologias. D. mexicana tiene un grado de parentesco mas alto, en terminos geneticos, con D. palus(n.sque con D.occiclentali.s. INTRODUCTION The genus Dirca L. is comprised of three species of slow-growing, understory shrubs found almost exclusively m nature on steep, westor north-facing slopes SIDA 21(2): 51 1-524. 2004 512 BRIT.ORG/SIDA 21(2) above a waterway (Johnson 1994; Nesom & Mayfield 1995; Graves 2004). While D. palusl lis L. is the most common of the species and is found in sparsely distributed colonies over most of eastern North America, the other two species, D. occidcnUilis Gray and D. mcxiaina Nesom & Mayfield, are rare and endemic. Di reel occidcntalis is isolated to a six-county region surroundmg the San Francisco Bay in Calilornia, and D. mexicanu is endemic to only one valley in the Sierra Madre Oriental Mountains of Tamaulipas, Mexico (Nesom r Graves 2004). Although there is interest in the genus due to the obscurity of plants in the wild, the vulnerability of plants m California (Johnson 1994), the discovery of the new species in Mexico (Nesom & Mayfield 1995), and the potential to utilize plants of this genus as shade-tolerant shrubs for managed landscapes (Dirr 1998), no research has been done to determine the phylogenetic relationships among the three species of Di rca. In the most recent treatment of the genus Dirca, Nesom and Mayfield (1995) showed the three disjuncts to be morphologically distinct and found that D. mcxicana more closely resembled D. oaulcntahs than it did D. paluslns. But, they also questioned the relative similarity of D. mcxiaina and D. Oiadcntalis "in view oi the well-known pattern of close relationship and disjunction between species of the eastern and southeastern United States and the sierra of northeastern Mexico" (Nesom & Mayfield 1995). Considering biogeographical trends, Nesom and Mayfield (1995) believed the best explanation for the origin of three disjunct species was, first, the isolation of the ancestors of D. occidentalis Irom those of D. palustns, and, more recently, the isolation of the ancestors of D. mcxicana trom those of D. palustris. Our goals were to resolve the apparent inconsistency between morphological and biogeographical evidence by examining the genotypic relationships among the three species of Dirca, to reconstruct the phylogeny of Dirca spp. by utilizing methods of molecular systematics, and to determine if molecular evidence supports the classification of D. mcxicana as a distinct species. We used two classes of molecular markers, Internal Transcribed Spacer (ITS) sequences and inter-Simple Sequence Repeats (ISSR), to quantify the genotypic variability ot Dirca. ITS techniques compare the internal transcribed spacer sequences of the 18S-5.8S-26S nuclear ribosomal DN A. They hold many advantages over other methods, including biparental inheritance, intergenomic variability suitable lor phylogenetic inference at the specific, generic, and family levels (Baldwin f992; Baldwin et al. 1995), and easy amplibcation with universal primers (White et al. 1990). ITS sequence data arc abundant and easily accessible in public databases, enabling direct comparisons among taxa and thus are used extensively tor botanical phylogenetics at generic and inlrageneric levels (Alvarez & Wendel 2003). ISSR techniques (Zietkiewicz et al. 1994) are chosen most often for their SCHRADER AND GRAVES, SYSTEMATICS OF DIRCA 513 capacity to resolve molecular differences below the specific level, but ISSRs are also valued because they sample a large portion of the genome and therefore avoid the bias accompanying phylogenies based on the sequence of only one or a few genes (Schrader & Graves 2004). Used together, these two methods can provide excellent resolution of genetic variability at and below the family level and proved effective for assessing infrageneric differences within the genus

Abstract: The genus Dirca L. (Thymelaeaceae) consists of three species of understory shrubs. Dirca palustris L. is sparsely distributed across eastern North America, D. occiden- talis Gray is endemic near the San Francisco Bay, and D. mexicana Nesom & Mayfi eld is known only in one population in northeastern Mexico. Despite interest in the horticultural use of Dirca, plants seldom are marketed. Diffi cult propagation impedes production of Dirca. We sought to defi ne protocols that promote uniform seed germination of all three Dirca spp. Endodormancy and paradormancy cause sporadic germination over several years under natural conditions, but endocarp removal, cold stratifi cation, and treatment with GA 3 increased germination percentage, speed, and uniformity. Dirca occidentalis was most responsive; up to 94% of seeds germinated after endocarp removal, 24 hours in GA 3 at 50 mg·L -1 , and stratifi cation at 4 °C for 30 days. Treatments also were effective for D. palustris (up to 68% germination), but seeds of D. mexicana were unresponsive and germinated at 25% or less. Seed treatments should facilitate production of D. occidentalis and D. palustris, but further research is needed to defi ne methods to propagate D. mexicana for horticultural use and for conserving this rare species in the wild.