Systematic Botany (2013), 38(1): pp. 82–91 © Copyright 2013 by the American Society of Plant Taxonomists DOI 10.1600/036364413X661971 Carex viridistellata sp. nov. (Cyperaceae), a New Cryptic Species from Prairie Fens of the Eastern United States Nathan J. Derieg,1,3 Sarah J. Weil,1,4 Anton A. Reznicek,2 and Leo P. Bruederle1,5 1Department of Integrative Biology, University of Colorado Denver, Denver, Colorado 80217–3364, U. S. A. 2University Herbarium, 3600 Varsity Drive, University of Michigan, Ann Arbor, Michigan 48108–2228, U. S. A. 3Present address: Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, California 93106, U. S. A. 4Present address: Department of Biological Sciences, Columbia University, New York, New York, 10027, U. S. A. 5Author for correspondence ([email protected]) Communicating Editor: Mark P. Simmons Abstract—Divergence between evolutionary lineages is not always marked by the development of obvious species-specific characters, whether morphological, physiological, or ecological. Consequentially, extant biodiversity can easily be overlooked. These cryptic species are often not recognized until genetic data are in hand, as is the case for the novel taxon we describe here. Carex viridistellata in Carex section Ceratocystis is an endemic species restricted to calcareous wetlands of Michigan, Ohio, and Indiana, where it has previously been collected as Carex cryptolepis. Crosses between Carex viridistellata and Carex cryptolepis produce sterile F1 hybrids, and the two species are differentiated by a number of subtle morphological characters, as well as aspects of their respective ecologies. Phylogenetic analyses of nrDNA strongly indicate monophyly of Carex viridistellata and its sister species relationship with the North Carolina narrow endemic Carex lutea. Both species constitute a distinct lineage within a generally poorly resolved section Ceratocystis. This work highlights the broad importance of cryptic taxa, with implications for fields ranging from population genetics to conservation and restoration. Keywords—Carex viridistellata, Cariceae, cryptic species, Cyperaceae, nrDNA, prairie fen. Carex L. is the largest genus in the sedge family (Cyperaceae), of section Ceratocystis in northern North America. Carex comprising approximately 2,000 species (Reznicek 1990). cryptolepis is distributed north of the Last Glacial Maximum Essentially cosmopolitan in distribution and occurring in a in eastern North America, being broadly sympatric with wide range of habitats, Carex is particularly well represented C. flava and C. viridula. Carex saxilitorralis Robertson was in temperate, boreal, arctic, and alpine wetland ecosystems described in 1980 from Atlantic Canada (Robertson 1980), (Ball and Reznicek 2002). This breadth of geography and ecol- but subsequently reduced to varietal status as C. viridula ogy generally lacks a concomitant diversity of phenotypes; the subsp. brachyrrhyncha var. saxilittoralis (Robertson) Crins (Crins determinant morphological differences between species can and Ball 1989b). Evolutionary relationships of the endemic be quite subtle. Even in North America, where the genus has North American taxa are uncertain. Crins and Ball (1989b) received a relatively large amount of attention resulting in suggested that C. cryptolepis diverged from C. flava, or a com- approximately 480 described species (Ball and Reznicek mon ancestor, in eastern North America, while Le Blond et al. 2002), new species continue to be described (e.g. Catling et al. (1994) tentatively proposed a close relationship between 1993; Hartman and Nelson 1998; Ertter 2000; Rothrock and C. lutea and C. cryptolepis. Reznicek 2001; Werier 2006; Wilson et al. 2007; Sorrie et al. Geographic extent of the other North American represen- 2011). Many of these species are cryptic (as defined in Bickford tatives of section Ceratocystis is great: C. flava and C. viridula et al. 2006), having been classified as previously described Michaux subsp. viridula var. viridula are broadly distributed species, until being recognized and segregated as distinct across Europe, Asia, and North America; while C. hostiana (e.g. Carex missouriensis P. Rothr. & Reznicek or C. shinnersii and several other subordinate taxa of C. viridula (ssp. P. Rothr. & Reznicek). Herein, we describe a species new oedocarpa and brachyrhyncha) have amphi-Atlantic distribu- to science that was discovered while undertaking research tions (Crins and Ball 1987; Crins and Ball 1989b). Variation addressing population genetic diversity and structure in for morphological, ecological, and cytological characters two North American endemics within Carex section Ceratocystis within these taxa is almost entirely shared between North Dumortier (Derieg et al. 2008). American and European populations, reflecting limited diver- Carex section Ceratocystis is monophyletic and closely related gence and a high degree of phenotypic plasticity (Crins and to Carex section Spirostachyae Drejer (Crins and Ball 1988; Ball 1987; Crins and Ball 1989a). Carex flava and C. viridula Escudero et al. 2008; Escudero and Lucen˜o 2009; Waterway Michaux subsp. viridula var. viridula might have spread into et al. 2009), in which the section has sometimes been sub- North America from Asia prior to the Pleistocene (Crins and sumed. Section Ceratocystis is one of the most well studied Ball 1989b). Kuchel and Bruederle (2000) found that allozyme groups of sedges worldwide, with numerous studies genetic diversity of North American populations of C. addressing cytogenetics; physiology, physiological ecology, viridula subsp. viridula var. viridula represents an extreme and ecology; taxonomy; population genetics; and systemat- subset of that found in European populations, as predicted ics. Of the five currently recognized species occurring in for a relatively recent, rapid migration into North America. North America (Crins 2002), two are endemic: C. cryptolepis A limited sampling of four North American populations of Mack. and C. lutea Le Blond. Carex lutea comprises eight C. flava (Derieg 2007) exhibits a similar pattern when com- populations restricted to the Cape Fear watershed in North pared to earlier allozyme-based population genetic studies Carolina, where it occurs in savannas on wet, sandy soils of European populations (Bruederle and Jensen 1991). overlying coquina limestone (Le Blond et al. 1994); as such, While conducting allozyme analyses on population sam- it is disjunct from the more or less continuous distribution ples collected from central and southern Ohio presumed to 82 2013] DERIEG ET AL.: CRYPTIC CAREX SPECIES NEW TO EASTERN NORTH AMERICA 83 be C. cryptolepis, a novel cryptic taxon was identified (Derieg following cycle sequencing with the GenomeLab DTCS quick start kit for et al. 2008). Using molecular genetic and morphological dye terminator cycle sequencing (Beckman Coulter, Fullerton, California). Forward and reverse sequence reads were edited and assembled into characters, we tested the hypothesis that populations of this contigs in Sequencher 4.5 (Gene Codes Corp., Ann Arbor, Michigan). putatively novel taxon represent an exclusive lineage that is Outgroup taxa were identified by BLAST searches of the NCBI nucle- genetically and morphologically distinct from other taxa otide database GenBank, as well as by examination of phylogenies in within Carex section Ceratocystis. We also tested the hypothe- Hendrichs et al. (2004), Escudero et al. (2008), and Escudero and Lucen˜o sis that the three North American endemic species of section (2009). Additional ITS and ETS sequences of taxa within Carex section Ceratocystis species were obtained from NCBI and included in the Ceratocystis form a monophyletic group. datasets. Datasets comprising the ITS-1, 5.8S, and ITS-2 region alone and the ETS 1f region alone were aligned in ClustalX version 2.0.11 using default alignment parameters (Thompson et al. 1997). A third dataset Materials and Methods was constructed by concatenating only those ITS and ETS sequences that were derived from a single individual; individuals with only one nrDNA Herbarium and Field Collections—Fieldwork was conducted across the region, and taxa where the fragments came from different individuals, extreme southern portion of the range of Carex cryptolepis in 2005 and were excluded from the analysis. Gaps that resulted from joining trun- 2006, during which population level collections of leaf tissue were cated sequences were filled with Ns. obtained and preserved appropriately for population genetic and phylo- Appropriate models of DNA substitution were tested for each data set genetic analyses, respectively (Derieg et al. 2008). Vouchers were taken by the program jModelTest version 0.1.1 (Posada 2008). Of the 88 tested for each individual sampled and representatives deposited in the models, the best was chosen by Akaike information criterion (AIC; Kathryn Kalmbach Herbarium (KHK) at the Denver Botanic Gardens. Akaike 1974). Phylogenetic analysis of each dataset using the model iden- For further morphological analyses and in order to determine the geo- tified by jModelTest was performed in the program PhyML version 3.0 graphical range of the putatively new taxon, herbarium accessions were starting from a BioNJ tree with optimal tree topologies estimated by subsequently examined from eight midwestern herbaria: Butler Univer- subtree pruning and regrafting (SPR) and branch lengths and substitu- sity (BUT), Indiana University (IND),