Systematic Botany (2013), 38(1): pp. 82–91 © Copyright 2013 by the American Society of Taxonomists DOI 10.1600/036364413X661971 viridistellata sp. nov. (), 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). 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; ; 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), University of Michigan (MICH), tion model parameters optimized (Guindon et al. 2005; Guindon et al. The Morton Arboretum (MOR), Michigan State University (MSC), Miami 2010). Nonparametric bootstrapping (Felsenstein 1985) with 1,000 repli- University (MU), Ohio State University (OS), and Ohio University (BHO). cates was performed to obtain a measure of support for each branch in Morphological Analyses—Measurements from pressed herbarium the maximum likelihood tree. Trees were viewed, edited, and exported specimens were made for 34 vegetative characters and 59 reproductive to images using the program FigTree version 1.3.1 (http://tree.bio.ed.ac characters (Table S1), using digital calipers for lengths less than five mm. .uk/software/figtree/). Fertile culm height (N = 20) was measured from the base of the plant to Hybridization and Species Limits—During the course of field and the tip of the uppermost spike, width (N = 17) was taken from the base of herbarium research, putative hybrids involving the new taxon were the lowest spike. Vegetative shoot height (N = 6) was measured from the observed at three sites in Michigan and Ohio. Cleaved amplified poly- base of the plant to the tip of the longest leaf, and pseudoculms (N = 6) morphisms (CAPs) (Konieczny and Ausubel 1993) were used to test the were measured from the base to the tip of the longest sheath. All leaves hypothesis of a hybrid origin at the Springville Marsh site. The CAPs (N = 17), including dead, brown leaves, were counted and the widest was analyses utilize a restriction enzyme to cleave amplified DNA fragments measured for width (N = 18). Ligule length (N = 12) was measured from in a species-specific manner, thereby yielding observable differently- the point of leaf attachment to the tip of the ligule. length sized fragments following PCR amplification, digestion, electrophoresis, (N = 19) was taken from the node of the lowest subtending bract to the tip and visualization. In addition to the three putative hybrids, we screened a of the most distal spike. Peduncle length (N = 16) was measured from the large number C. cryptolepis from the population to determine whether node of the subtending bract to the base of the lowest flower. Subtending introgression of C. viridistellata nrDNA had occurred. We did not observe bract length (N = 15) measurements included only the free portion of the C. viridistellata during our collections, although herbarium records indi- bract and width (N = 17) was measured from the widest point. The cate it does occur in the area. number of staminate spikes (N = 19) was counted from a single fertile Preliminary ITS, ETS, and plastid sequence data were screened for culm, and staminate spike dimensions were taken from the widest (N = 20) polymorphisms and NEB cutter V2.0 (New England Biolabs, Ipswich, and longest points (N = 19). The number of pistillate spikes (N = 20) was Massachusetts) was used to identify species-specific restriction enzyme counted from a single fertile culm, and pistillate spike dimensions were cut sites. Enzymes were chosen based on their ability to differentially taken from the widest and longest points (N = 19). When a distal pistillate cut PCR-enriched nrDNA fragments obtained from C. viridistellata, C. spike was present, the distance between the lowest two spikes was mea- cryptolepis, and their putative hybrids: Hinf I and MslI (nrDNA, ETS); sured (N = 2). Number of scales on staminate spikes (N = 16) and Eco0109I, BccI, and BsaHI (nrDNA, ITS); and AluI (cpDNA, trnL-trnF). perigynia on pistillate spikes (N = 17) were estimated from one side of Nuclear ribosomal DNA regions were PCR amplified as above, while the spike. Staminate scale (N = 18), perigynium (N = 19), and achene reaction conditions were modified for the cpDNA region: final concentra- widths (N = 17) were taken from the widest point. Beak length (N = 19) tion of MgCl2 was 3 mM; and final concentration of each primer was was measured from the apex of the angle formed between the beak and 0.4 mM (TabC and TabF, Taberlet et al. 1991). Thermal cycler conditions the base of the perigynium to the tip of the beak. These data were ana- for all regions were the same as above. lyzed to determine range, mean, and standard deviation. Digestions were performed at a final volume of 20 ml with 50–100 ng/ml Phylogenetic Analyses—DNA was isolated from leaf tissue, either PCR-amplified DNA, 2 ml of 10 + buffer (contents vary with enzyme), silica-gel dried field-collections or pressed specimens, using the DNeasy 0.1 mg/ml BSA (when required by a specific enzyme), and 0.5–5 units plant mini kit (Qiagen, Valencia, California) or following Dellaporta et al. enzyme, brought to volume with de-ionized water. Appropriate buffer (1983). Nuclear ribosomal DNA regions were amplified in 25 mL total- and BSA were supplied with the enzyme from New England Biolabs. volume reactions composed of: 2.5 mL 10 + reaction buffer; 1.5 mL 25 mM Digestions were incubated at 37C for 2–6 hrs and visualized on a 1.5% MgCl2; 1.0 mL 25 mM dNTPs, equimolar ratio; 0.25 mL 1 U/mL Taq poly- agarose gel containing 0.33 mg/ml ethidium bromide. All digests merase; 1.5 mL 10 mM forward primer; 1.5 mL 10 mM reverse primer; 1 mL included an uncut control. of 10 ng/mL sample DNA; and 15.75 mL water. The 5.8S ribosomal  subunit plus the flanking internal transcribed spacer regions (ITS-1 and ITS-2) were amplified as a single fragment using primers ITS 4i and ITS 5i Results (Roalson et al. 2001). A portion of the 50 end of the external transcribed spacer (ETS 1f) was amplified using primers ETS1f and 18Sr (Starr et al. Phylogenetic Analyses—We generated nrDNA fragment 2003). Both fragments were amplified using identical thermal cycler con- sequences for 33 individuals from Carex section Ceratocystis: ditions: an initial denaturation at 95C for 2 min; followed by 32 cycles of denaturation (95C for 1 min), primer annealing (55C for 30 s), and both ITS and ETS for 20 individuals; ETS for 11 individuals; strand extension (72C for 1 min); and a final extension at 72C for 10 min. and ITS for two individuals (Table 1). Ingroup sequences Amplification products were prepared for dye-terminator cycle from a previous study (Derieg et al. 2008) were also included, sequencing by ExoSAPIT (USB, Cleveland, Ohio) treatment, ethanol pre- cipitation, and re-suspension in molecular grade water. Sequencing was adding an additional 16 individuals with both ITS and ETS performed at the Rocky Mountain Center for Conservation Genetics and sequences and one individual with only ETS. A search of the Systematics on a Beckman Coulter CEQ 8000 genetic analysis system, NCBI nucleotide database provided ITS sequences for an 84 SYSTEMATIC BOTANY [Volume 38

Table 1. Nuclear ribosomal DNA sequences used for phylogenetic analyses of Carex section Ceratocystis. Sequences either previously published or downloaded from GenBank are indicated as such in the “Source” column.

Accession numbers

Taxon Synonym Sample Country Province/State ETS ITS Source C. cryptolepis 604 U. S. A. Michigan JX409819 JX409850 C. cryptolepis FR29 Canada Ontario EU247849 EU247866 Derieg et al. 2008 C. cryptolepis IP37 U. S. A. Ohio EU247850 Derieg et al. 2008 C. cryptolepis IR01 U. S. A. New York JX409820 JX409851 C. cryptolepis ML08 U. S. A. Wisconsin EU247851 EU247868 Derieg et al. 2008 C. cryptolepis MN0220 U. S. A. Minnesota EU247852 EU247869 Derieg et al. 2008 C. cryptolepis SB21 U. S. A. New York EU247853 EU247870 Derieg et al. 2008 C. cryptolepis TP03 U. S. A. Maine JX409839 C. flava BBFb04 Canada Newfoundland EU247856 EU247873 Derieg et al. 2008 C. flava BBFb06 Canada Newfoundland JX409821 JX409852 C. flava BBFb34 Canada Newfoundland JX409822 JX409853 C. flava GL35 Canada Ontario JX409840 C. flava C. flava var. alpina GS17 Austria JX409823 JX409854 C. flava GV25 Norway JX409824 JX409855 C. flava LE14 Switzerland JX409825 JX409856 C. flava RT03 Sweden EU247857 EU247874 Derieg et al. 2008 C. flava RT08 Sweden JX409826 JX409857 C. flava RT11 Sweden JX409827 JX409858 C. flava SB04 U. S. A. New York JX409828 JX409859 C. flava C. flava var. alpina TH16 Austria JX409841 C. flava WB06 U. S. A. Vermont JX409842 C. flava WB12 U. S. A. Vermont JX409843 C. flava WFB16 U. S. A. Virginia EU247858 EU247875 Derieg et al. 2008 C. flava Germany AY278310 GenBank C. flava Canada Quebec AY757596 GenBank C. flava Russia AF285007 GenBank C. flava C. nevadensis Spain DQ384172 GenBank C. flava Spain DQ384144 GenBank C. flaviformis New Zealand AY699610 GenBank C. hostiana Switzerland EU288555 GenBank C. hostiana MA Canada Quebec JX409829 JX409860 C. hostiana REZ Canada JX409830 JX409861 C. hostiana France AY278309 GenBank C. lutea HR07 U. S. A. North Carolina EU247861 EU247878 Derieg et al. 2008 C. lutea HR26 U. S. A. North Carolina EU247862 EU247879 Derieg et al. 2008 C. lutea NS19 U. S. A. North Carolina EU247863 EU247880 Derieg et al. 2008 C. lutea OB36 U. S. A. North Carolina EU247864 EU247881 Derieg et al. 2008 C. lutea PL08 U. S. A. North Carolina JX409844 C. lutea SC18 U. S. A. North Carolina JX409845 C. viridistellata AF07 U. S. A. Ohio EU247859 EU247876 Derieg et al. 2008 C. viridistellata IMI38 U. S. A. Indiana JX409846 C. viridistellata LQ32 U. S. A. Ohio EU247860 EU247877 Derieg et al. 2008 C. viridula Canada Quebec AY757597 GenBank C. viridula Canada AY278308 GenBank C. viridula Switzerland AY278290 GenBank C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa BBFa02 Canada Newfoundland JX409831 JX409862 C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa BBFa14 Canada Newfoundland EU247854 EU247871 Derieg et al. 2008 C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa BBFa30 Canada Newfoundland JX409832 JX409863 C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa CS09 Switzerland JX409870 C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa GS10 Austria EU247855 EU247872 Derieg et al. 2008 C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa Spain DQ384164 GenBank C. viridula subsp. brachyrrhyncha var. elatior C. lepidocarpa Germany AY278293 GenBank C. viridula subsp. oedocarpa C. demissa DT14 Germany JX409833 JX409864 C. viridula subsp. oedocarpa C. demissa HR30 Norway JX409847 C. viridula subsp. oedocarpa C. demissa Spain DQ384119 GenBank C. viridula subsp. oedocarpa C. demissa Germany AY278307 GenBank C. viridula subsp. viridula var. viridula AL25 U. S. A. JX409835 JX409865 C. viridula subsp. viridula var. viridula Asa10 Sweden JX409848 C. viridula subsp. viridula var. viridula Asa19 Sweden JX409834 JX409866 C. viridula subsp. viridula var. viridula GL37 Canada Ontario EU247865 EU247882 Derieg et al. 2008 C. viridula subsp. viridula var. viridula GL39 Canada Ontario JX409836 JX409867 C. viridula subsp. viridula var. viridula HF03 U. S. A. Colorado JX409837 JX409868 C. viridula subsp. viridula var. viridula KC16 U. S. A. Washington JX409871 C. viridula subsp. viridula var. viridula OW05 U. S. A. Ohio JX409849 C. viridula subsp. viridula var. viridula RL01 Canada Ontario JX409838 JX409869 C. cretica DQ384116 GenBank C. cretica DQ384117 GenBank C. cretica DQ384118 GenBank

(Continued) 2013] DERIEG ET AL.: CRYPTIC CAREX SPECIES NEW TO EASTERN NORTH AMERICA 85

TABLE 1. Continued.

Accession numbers

Taxon Synonym Sample Country Province/State ETS ITS Source C. densa EU000962 GenBank C. densa EU000963 GenBank C. diluta DQ384120 GenBank C. distans DQ384127 GenBank C. distans DQ384126 GenBank C. echinochloe AY241993 AY241992 GenBank C. extensa DQ384128 GenBank C. extensa AY278311 GenBank C. pendula AY757661 AY757600 GenBank C. punctata AY757659 AY757598 GenBank C. sylvatica AY757660 AY757599 GenBank C. sylvatica AY278306 GenBank

additional 15 individuals representing ingroup taxa. Sequences monophyly of these five North American populations of for outgroup species were also obtained from NCBI, con- C. flava to the exclusion of European samples increased to sisting of four accessions with both ITS and ETS and 11 with 98%, although this could be attributed to samples with only only ITS. An anonymous reviewer suggested that the NCBI one sequenced nrDNA region excluded from the analysis. accessions AY241993 and AY241992, for C. echinochloe ITS Carex cryptolepis had weak support for monophyly. The and ETS sequences, possibly came from a misidentified spec- joined ETS-ITS tree resolved C. cryptolepis with only 57% imen of C. mannii; herein, we have left the accessions as they bootstrap support (unresolved polytomy in the ETS tree, are annotated on GenBank, as the actual identity of the sam- and paraphyletic and polyphyletic in the ITS tree, where ple will not affect any conclusions regarding relationships the New Zealand species C. flaviformis, as well as multiple within Carex section Ceratocystis. The ITS alignment included C. viridula accessions fall into the paraphyletic C. cryptolepis). 68 sequences and 616 characters, the ETS alignment included Hybridization and Species Limits—Restriction digests of 52 sequences and 598 characters, and the joined ETS-ITS ETS nrDNA amplified fragments for three putative hybrids alignment had 40 sequences and 1,214 characters; gaps were from the Springville Marsh population yielded a combined treated as missing data. For ETS, ITS, and ETS-ITS the AIC- banding pattern representing both parental species (Fig. 2a). chosen DNA substitution models were, respectively, GTR + G, Results for ITS similarly demonstrated combined parental TIM2 + I, and GTR + G. banding patterns for the three hybrids, although the CAP Strong support for a monophyletic Carex section Ceratocystis banding patterns for ITS did not exactly match the predic- was seen, with the section being relatively highly differenti- tions from in silicio digestions. There was no evidence of ated from outgroup taxa in both the ITS (Fig. S1) and ETS introgression, as each of the additional 47 assayed individ- trees (Fig. S2) (96 and 100% bootstrap support, respectively). uals from this population exhibited ETS and ITS CAPs The joined ETS-ITS tree (Fig. 1) also placed taxa into a mono- banding patterns characteristic of C. cryptolepis. ITS bands phyletic section Ceratocystis (100% bootstrap support). Carex were often faint compared to ETS bands. viridistellata appeared as monophyletic in all analyses, con- The CAP banding pattern for the trnL-trnF cpDNA ampli- sistently resolved as the sister species to Carex lutea; boot- fied fragment matched that of C. cryptolepis (Fig. 2b), lacking strap support for the sister relationship was weaker in the the additive pattern seen in the nuclear sequences. A 1,048 bp ITS tree (66% bootstrap support) than the ETS tree (91% band, corresponding to the uncut sequence size, occurred in bootstrap support) or the joined tree (91% bootstrap sup- all of the digestions. This band was diminished, but not elim- port). Weak support for a three species clade comprising the inated after increased enzyme concentration and digestion C. viridistellata and C. lutea sister pair and a basal C. hostiana time (data not shown). was seen in the ETS tree (56% bootstrap support), but was not seen in the ITS tree, where C. hostiana constitutes a basal Discussion grade in the section. The C. hostiana/C. viridistellata/C. lutea clade was recovered in the joined ETS-ITS tree, but boot- The novel taxon described herein, which we have called strap support was less than 50%. A monophyletic clade of Carex viridistellata, is a distinct species within Carex section C. cryptolepis, C. flava, and C. viridula (and subspecific taxa) Ceratocystis, endemic to undisturbed calcareous wetlands of was recovered with strong support in the joined ETS-ITS tree Michigan, Indiana, and Ohio (Fig. 3). While rare across this (93% bootstrap support) and weak support in the ETS tree distribution, C. viridistellata can be abundant locally and even (52% bootstrap support). a dominant species in open areas of some sites. Sites are Relationships of Carex cryptolepis, C. flava, and the sub- characterized by wet, sandy substrate, frequently overlying specific taxa of C. viridula were poorly resolved in all ana- limestone bedrock (e.g. Lynx Prairie, Ohio) or with visible lyses. A clade of predominantly North American C. flava was marl (e.g. Ankeney Fen, Ohio) and are always found in seen in both the ITS and ETS trees (88% and 51% bootstrap sunny, open areas; in these aspects of ecology, C. viridistellata support, respectively), but not all North American samples is much like its sister species C. lutea, a calciphile intolerant of are nested within it; in both trees, the clade included a Euro- shrub or tree canopy (Le Blond et al. 1994). Plants are robust pean accession, although which accession differed between and can be large for members of section Ceratocystis, trees. In the joined ETS-ITS tree (Fig. 1), support for the approaching the size of C. lutea; we have observed in both 86 SYSTEMATIC BOTANY [Volume 38

Fig. 1. Maximum likelihood tree for joined ETS-ITS nrDNA sequences with percent bootstrap support indicated for nodes with greater than 50% support. Accession number or sample name is listed after the species name. Dark grey boxes mark monophyletic clades of North American accessions. species that the largest individuals occur in locations receiv- suggests) and larger individuals can overlap to a small extent ing less light, e.g. at the transition between prairie opening in size characteristics (Crins and Ball 1989b). Like C. lutea, the and woods (C. viridistellata at IMI Fen, Indiana). perigynium beaks of C. viridistellata are typically sparsely sca- Although initially recognized on the basis of allozyme brous (Fig. 4), whereas they are always smooth in C. cryptolepis; genetic differentiation, including novel alleles, C. viridistellata a minute’s time with a hand lens easily makes the distinction. can be discriminated from other species in the section on the Probably due to ecological differentiation, C. viridistellata basis of a number of discernible, albeit subtle, morphological does not appear to frequently co-occur with the species with characters. Carex lutea has shorter perigynium beaks, but which it is marginally sympatric (C. cryptolepis, C. flava, and confusion between the species is unlikely given the large C. viridula); it is, however, syntopic with C. cryptolepis at disjunction of approximately 700 km (Le Blond et al. 1994). Springville Marsh in central Ohio, where we have documented Carex flava has darkly pigmented pistillate scales clearly C. cryptolepis + viridistellata hybrids, and possibly other his- standing out against the perigynia, while C. viridula is a much toric sites. Cleaved amplified polymorphism (CAP) banding smaller plant overall, particularly with respect to perigynium patterns for nrDNA fragments from hybrid, C. viridistellata and beak length (Crins and Ball 1989b). In the field, confusion and C. cryptolepis individuals from Springville Marsh con- with C. cryptolepis is most likely, as C. cryptolepis has pistillate firmed the morphologically based identification of these scales that are similar to C. viridistellata (i.e. almost indistin- hybrids. The maternal plant in each case of hybridization was guishable from the perigynia, as the specific epithet “cryptolepis” C. cryptolepis, as determined by CAPs in the trnL-trnF cpDNA 2013] DERIEG ET AL.: CRYPTIC CAREX SPECIES NEW TO EASTERN NORTH AMERICA 87

Fig. 2. Cleaved amplified polymorphisms of ETS nrDNA (A) and trnL-trnF plastid (B) fragments for Carex cryptolepis, C. viridistellata, and hybrids from Springville Marsh, Ohio.

Fig. 3. Distribution of Carex viridistellata documented from herbar- region. Although our small sample of hybrid individuals does ium records and field collections. not allow for a statistical test of the directionality of pollen movement during hybridization between C. cryptolepis and evolutionary divergence when compared to any species pair C. viridistellata, our results are consistent with observations in the C. flava complex. from other plant families where the maternal plant of hybrids Phylogenetic analyses of nrDNA regions (ETS and ITS) is often the species having a shorter style (e.g. Kiang and provide strong support for a sister species relationship Hamrick 1978; Williams and Rouse 1988; Field et al. 2011). between C. viridistellata and C. lutea, together constituting a However, Cayouette and Catling (1992) point out that when distinct lineage within section Ceratocystis (Figs. S1, S2, and hybrids are found in an area with only one parental species, as 1). Carex section Ceratocystis as a whole has been recovered as ours were, it is often the maternal species; as such, we cannot monophyletic by other researchers (Hendrichs et al. 2004; say whether the apparent directionality of gene flow between Escudero et al. 2008; Escudero and Lucen˜o 2009), and this species is caused by pollen tube and style length mismatch is a strongly supported result in our analyses as well. Carex or is simply a consequence of local demographics. cryptolepis, C. flava, and C. viridula form a monophyletic group Based on morphology, a C. cryptolepis + viridistellata hybrid that has been recognized previously as the C. flava complex. was documented from Brown’s Lake in Jackson Co., Relationships within the C. flava complex are poorly Michigan (MSC), and C. cryptolepis + viridistellata and C. resolved, these difficulties are likely an issue of the evolu- viridula + viridistellata hybrids were documented from a site tionary history of nrDNA and its limited power to resolve near Bayport in Huron Co., Michigan (MICH). phylogenetic relationships at this level in the genus, rather Introgression between C. viridistellata and C. cryptolepis is than an issue of taxon sampling. Introgression, incomplete likely rare or non-existent, considering all of the hybrids we lineage sorting, and limited sequence divergence could all examined were sterile (no fully developed achenes were be contributing to this problem. Placement of C. hostiana is found) and nrDNA CAPs from a larger survey of individuals also uncertain, and we cannot make any conclusion about of C. cryptolepis in the population failed to recover any var- which of the possible topologies is correct: sister to the iation from the predicted banding pattern. Although intro- C. flava complex, sister to C. lutea/C. viridistellata, or ancestral. gression has been documented between other species in Crins and Ball (1988) described several evolutionary trends the section (e.g. Blackstock and Ashton 2010), the lack of in section Ceratocystis, where more derived taxa are smaller, introgression between C. viridistellata and C. cryptolepis is not shorter lived, and quicker to reproduce; have higher chro- especially surprising considering their relatively greater mosome counts; and have increasingly better defined silica 88 SYSTEMATIC BOTANY [Volume 38

Fig. 4. Carex viridistellata. A. Habit. B. Perigynium, dorsal and lateral surface views, respectively. C. Pistillate scale, dorsal and ventral surface views, respectively. D. Achene. E. Inflorescence. bodies in achene epidermal cells. We do not have these data from an ancestral state of calciphily. Our observations of field for C. viridistellata but, considering our phylogenetic ana- sites where C. viridistellata occurs suggest that it retains the lyses, predict the species to generally exhibit character states ancestral calciphily. more towards the ancestral end of such trends. Just consider- Pleistocene glaciations undoubtedly had a major impact ing overall plant size, C. viridistellata is more like C. hostiana on the recent evolutionary history of C. viridistellata. Most or C. flava than C. viridula. Another evolutionary pattern populations of the species occur north of the Last Glacial described by Crins and Ball (1989a) is that calcifugy (e.g. in Maximum, except for those in the Nature Conservancy’s C. cryptolepis) has evolved multiple times independently Edge of Appalachia Preserve in Adams Co., Ohio. Across 2013] DERIEG ET AL.: CRYPTIC CAREX SPECIES NEW TO EASTERN NORTH AMERICA 89 five populations for which we have data, including one in Plants caespitose, fertile culms 12.9–68.1(–82.3) cm tall, Edge of Appalachia that likely persisted locally through 0.6–1.3 mm wide at base of lowermost spike, trigonous, glaciation events, C. viridistellata maintains no polymorphic green, glabrous with occasional scabrous edges near spikes. loci at either the population or species level (Derieg et al. Leaves 1–13; basal usually with one cauline; blades and 2008; Derieg and Bruederle unpubl. data). It is likely that sheaths (4.4–)6.3–70.2(–74) cm tall, widest blades 1.9–3.3 mm C. viridistellata experienced multiple cycles of range expan- wide; green, occasionally yellowish, often with brown or yel- sion and contraction in response to Pleistocene glacial- low tips; heavily scabrous on apical, abaxial margins and interglacial cycles, and such cycles can stochastically purge veins. Sheaths 2.5–38.0(–49.8) cm tall, glabrous, pale green genetic diversity through genetic drift. The resulting depau- to stramineous, and tightly adhering to the culm. Inner band perate allozyme genetic diversity of C. viridistellata is espe- of sheaths white and transparent, thin, concave, with round cially striking when one considers that the sister species, or truncate apex. Ligules round or obtuse, 1.3–2.4 mm; free C. lutea, maintains the highest levels of genetic diversity portion entire, white and transparent, 0.2–0.6 mm in length. observed in North American populations of section Ceratocystis Vegetative culms with mostly 7–14 leaves; leaves 2.1–59.5 cm taxa, comparable to levels of diversity seen in European tall, 1.9–3.6 mm wide, similar in color and texture to fertile representatives of the section (Bruederle and Jensen 1991; culm leaves; pseudoculm 2.5–11.1 cm tall. Kuchel and Bruederle 2000; Hedre´n 2002; Derieg et al. 2008; (1.5–)2.0–11.0 cm long or 19.8–29.2 cm when distal spike Blackstock and Ashton 2010). present. Staminate spikes terminal and singular, rarely with Cryptic species are a major concern in many biological 2 spikes; 6.0–23.8 mm long, 1.5–2.8 mm wide; peduncles fields and have been described from across the tree of life, 2.1–33.6 mm long, trigonous, often with lightly scabrous although vascular plants are under-represented in the litera- edges; ratio of peduncle length to spike length 0.2–2.1(–3.4). ture (Bickford et al. 2006). Difficult genera such as Carex may Staminate flowers ca. 30–98 per spike; scales lanceolate-ovate, be especially likely to harbor unrecognized species diversity acute or occasionally acuminate, 1(–3)-nerved, 3.2–4.5 mm due to unfortunate lumping of distinct evolutionary lineages long, 0.8–1.3(–2.0) mm wide with green center and thick in the absence of sufficient efforts to delimit species bound- white or brown transparent margins. Staminate spikes often aries. Carex viridistellata is a species new to science, which subtended by a scale-like bract, 2.7–10.3(–18.0) mm long, was initially identified on the basis of molecular genetic with scabrous central nerve, sheathing base, and transparent data, but subsequently found to differ morphologically, eco- margins. Pistillate spikes lateral, 1–3(–4), one spike per node; logically, and geographically from C. cryptolepis. There are ca. 40–96-flowered, occasionally with 5–10 staminate flowers two important conservation considerations arising from this at apices; ellipsoid or globose, (6.6–)7.23–18.8 mm long, research: first, C. cryptolepis is absent from Indiana and rarer (7.3–)10–13.5(–14) mm wide, length to width ratio (0.7–) than previously thought in Ohio, as many reported popu- 0.9–1.6(–1.7) but most often greater than one. When distal lations are actually C. viridistellata; and second, C. viridistellata spike present, distance between lowest spikes 36.7–262.0 mm. is a rare endemic with a limited distribution and high degree Peduncles of uppermost pistillate spikes sessile to 5.6 mm, of habitat specificity. lowermost spike peduncles 2.6–27.7(–33.2) mm long or 46.2– 103.0 mm long when distal spike present; trigonous with scabrous edges. Pistillate spikes subtended by leaf-like bracts, Taxonomic Treatment lower bracts ascending; lower blades 2.5–21.9(–24.1) cm long, Carex viridistellata Derieg, Reznicek, & Bruederle, sp. nov.— 1.5–2.8 mm wide, sheaths (1.0–)1.6–20.6 mm long; ratio of TYPE: U. S. A. Michigan: Washtenaw Co., ca. 5 miles lower bract length to inflorescence length (1.4–)1.6–3.2(–3.9) NNW of Dexter. Rich fen SE of Little Portage Lake dom- or (0.6–0.8)1.2–4.3 when distal spike present. Perigynia reflexed inated by Carex sterilis, Cladium mariscoides, Eleocharis or spreading, irregularly trigonous, glabrous, 9–13 nerved, rostellata, and forbs; abundant and robust, very locally 4.5–6.7 mm long; bases inflated, yellow to green, transparent, a dominant; NW1/4 sect. 12, T1S R4E; 4224046.300N, 0.8–2.1 mm wide; beaks green, angled or occasionally 8355020.100W, 1 July 2007 (fr), A.A. Reznicek 11873 (holo- straight, often with lightly scabrous margins, 2.0–3.2 mm long, type: MICH!, isotypes: CU!, DAO!, DBG!, F!, GH!, MO!, bidentulate; teeth white or light green, 0.3–0.8 mm long. Pis- MOR!, MU!, NY!, OS!, US!). tillate scales triangular-ovate, acute or acuminate, 1-nerved with green center and wide white or yellow transparent mar- Plantae cespitosae, culmi 12.9–68.1(–82.3) cm alti. Folia 1– gins, 2.2–3.5(–3.9) mm long, 0.6–1.3 mm wide and obscured 13; basalia plerumque una caulina; laminae vaginaeque by perigynia. Achenes trigonous, obovate, truncate; dark (4.4–)6.3–70.2(–74) cm altae, laminae 1.9–3.3 mm latae; brown when mature, light brown with cream edges when ligulae obtusae vel rotundatae, 1.3–2.4 mm longae. immature; 1.2–1.6 mm long and 1.0–1.3 mm wide at widest Inflorescentiae (1.5–)2.0–11.0 cm longae; spica staminata point. Figures 3, 4. terminali, pedunculo 2.1–33.6 mm longo, spicis pistillatis Phenology—Plants flower in the spring of the year, May or 1–3(–4), lateralibus, ellipsoidalis vel globosis, bracteatis; early June depending upon the latitude, micro-site, and bracteae foliacea, laminis 2.5–21.9(–24.1) cm longis, 1.5– season, with fruit maturation in July. 2.8 mm latis, vaginis (1.0–)1.6–20.6 mm longis. Perigynia Ecology and Distribution—Carex viridistellata is a broad patentia vel reflexa, irregulariter trigona, glabra 4.5–6.7 mm endemic that is sparsely distributed throughout its range in longa, 0.8–2.1 mm lata, basi inflata, lutea vel viridia, translucens; Michigan, Indiana, and Ohio (Fig. 3). It extends from Bay rostra 2.0–3.2 mm longa, viridia, curvata, marginibus saepe County, Michigan south to Adams Co., Ohio and west to scaberulis. Squamae pistillatae 2.2–3.5(–3.9) mm longae, 0.6– Berrien Co., Michigan and Wabash Co., Indiana. It is known 1.3 mm latae, ovati-triangulares, mediae viridiae, ad marginem from 20–25 sites historically, many of which are currently late albo- vel luteo-hyalinae. Achenia 0.2–1.6 mm longa, protected. It typically occupies a variety of wet, open habi- 1.0–1.3 mm lata, trigonia, obovata, truncata, brunnea. tats, with sandy soils that are typically rich in calcium. These 90 SYSTEMATIC BOTANY [Volume 38 range from prairie openings on wet, sandy soils overlying 2000, Gardner 3246 (OS); 1.47 mi NNE of jct Blacks Run and Waggoner limestone bedrock to sparsely vegetated marly seeps in fens. Riffle Rd., 1.34 mi ENE of jct Beasely Fork and Waggoner Riffle Rd., 1.34 mi ENE of BM 551, Green TWP, Concord Quad, 16 Jun 1999, The number of individuals found in a population is often Gardner 2542 with Gray (OS); Buzzardroost Rock Preserve, 0.4 mi NNW few, but can range from fewer than 25 individuals to 1,000 Twp Rt 152 and 0.55 mi NW Mt. Armenia Church, Brush Creek Twp, or more; in most cases, population extent is not great and 384504800N, 832603200W, 31 Jul 1981, Cusick 21,014 et al. (MU, OS); plants are consequently closely spaced. 1,100 ft NE of Weaver Rd jct with SR 125, 1,900 ft NW of Lynx Rd Etymology—The specific epithet “viridistellata” refers to junction with SR 125, Brush Creek Township Lynx Quad, 27 Jun 2000, Gardner 3302 (MU); ca. 1.6 mi E of the town of Lynx on S. R. 125, grow- the pistillate spikes, which are green (viridi) with perigynia ing in moist sandy soil, Brush Creek Twp., Lynx Quad., 25 Jul 1990, radiating like the rays of a star (stellata). Baird 983 (MU); Greene Co., Ankeney Fen, 3944033.6600N, 840022.6800W, Additional Specimens Examined—U. S. A. Indiana: Wabash Co., 20 Jun 2005 Derieg s. n. (DBG); Ankeney Fen, 1.0 mi. E. of Beaver Valley Round Lake at Laketon, 17 Sept 1916, Deam 22,002 (IND, NY); Henry Rd., 0.75 mi. W. of Trebein Rd., W. side of Beaver Creek, Sec. 16, Co., IMI Fen, 403046.1700N, 8521042.3100W, 28 Jun 2006, Derieg s. n. Beavercreek Twp., Bellbrook Quad., 27 May 1988, Stine 136 (MU); (DBG). Michigan: Bay Co., Bay City, 17 Jun 1902, Bradford s. n. (MSC); Ankeney Fen, 1.0 mi. E. of Beaver Valley Rd., 0.75 mi. W. of Trebein Berrien Co., Niles, 10 Jun 1939, (BUT); Huron Co., Bayport, 25 Jul 1951, Rd., W. side of Beaver Creek, Sec. 16, Beavercreek Twp., Bellbrook Hebert s. n. (MICH); T16N R09E Sec 21, 43.79261N, 83.41829W, 8 Jul Quad., 26 Jul 1988, Stine 209 (MU); Seneca Co., adjacent to boardwalk O’Connor 12 2008, (MICH); Jackson Co., along Swains Lake Drain, ca. about 500 feet from the entrance to Springville Marsh Nature Preserve, 3 mi S of Concord, NW 1/4 sect 9, T4S R3W, 9 Jul 2002, Reznicek 11357 south side of T 24, 1 mile west of US 23, Sec 31.; Big Spring Twp., with Noode´n (MICH); Kalamazoo Co.: Shore of Paw Paw Lake, Alvada Quad., 41 0 15.46 N, 83 24 5.06 W, 22 Jun 1993, Schneider 42.1656 N, 85.7472 W, 5 Jul 2008, Hamm s. n. (MSC); E. Shore of  0 00  0 00   1993:241 with Barber & Cochran (OS); southeastern corner of Springville Paw Paw Lake, off Paw Paw Lake Drive, 42 09 51 N, 85 44 56.8 W,  0 00  0 00 Marsh State Nature Preserve along the west side of the RR tracks. Sec. 15 Jul 2009, Reznicek 11981 with Hamm & Springer (AUB, MICH, MOR, 32. Big Spring Twp., Carey Quad., 29 Jun 1994, Schneider 1994:60 with NY, WMU); Lenawee Co., Goose Creek Fen, E. Woodstock Rd., 0.3 mi NE Windus, Cochrane, & Barber (MU); Springville Marsh State Nature Pre- of Little Goose Lake, 42.0632N, 84.3204W, 10 Aug 2009, Walters 1097 with Erskine, (MICH); Livingston Co., NE 1/4 section 2, T1N R5E, serve, S side of T24 1 mi. W of U. S. 23, NE 1/4 sect. 31, Big Spring Twp., Alvada Quad., 31 May 1990, McCormac, 2511 (MU); Springville Marsh ca. 2 1/2 mi. SW of Brighton, 4230050.900N, 8348058.800W, 5 Jul Nature Preserve, 3 1/2 mi. S., Alvada, Big Spring Twp, Alvada Quad., 2007, Reznicek 11874 (MICH); Washtenaw Co., T04S R03E Sec 6, 42.15511N, 28 May 1980, Brandenburg 402 (MU); Springville Marsh State Nature 84.13136W, 25 Jun 2008, O’Connor 10 (MICH); ca. 5 miles NNW of Dexter, fen SE of Little Portage Lake, NW 1/4 sect 12, T1S R4E, 13 Jun Preserve, along boardwalk, 0.175 mile due south of Twp. Rd. 24 (Muck 1991, Reznicek 8799 with Ludwig & Kadlec (MICH); 1 mile northwest of Road) and 0.25 mile due west of Chesapeake Ohio RR tracks, Alvada Four Mile Lake, 26 Jun 1941, Ludwig 1369 (MICH); Portage Lake 5 Jul Quad, Big Spring Township; common on sedge meadows, 5 June 1986, 1935, Hermann 6841 (MICH); Portage Lake, 8 Jul 1921, Walpole (MSC). Bissell 1986:091 with Bartlett & Danielson (MICH, CLM); Springville OHIO: Adams Co., near Lynx, 10 Jun 1939, Bartley s. n. (BHO); Cline Marsh State Nature Preserve along north side of boardwalk, 0.23 mile Road, 3845037.5600N, 8324013.7200W, 19 Jun 2006, Derieg s. n. (DBG); due west of Chesapeake and Ohio railroad tracks and 0.2 mi south of Lynx Prairie, 3845049.1500N, 8324039.2300W, 19 Jun 2006, Derieg s. n. Township Rd 24, Alvada Quad, Big Spring Twp.; occasional within (DBG); east of Lynx, 7 Aug 1961, Braun s. n. (OS); valley N. of Blacks western section of meadow, 15 Jul 1999, Bissell 1999:08 with Chirdon, Run rd. at mouth of Cave Hollow, 1 mi NE, Waggoner Riffle Rd. Green Punwani, & Bartlett, (MICH, CLM); Wyandot Co., remnant of Big Spring Twp., Concord Quad, 29 Jun 1978, Cusick 18469 (OS); 200 ft SSW of Jct of Prairie, NW 1/4 Sec. 5, T1S, R13E, Crawford Twp., ca. 2.5 mi. N of Wilderness Road and SR 125, Brush Creek Twp, LYNX QUAD, 20 Jun Carey, 4059022.3800N, 8323057.6700W, 12 Sep 1969, Stuckey 8430 (OS).

Key to Carex section Ceratocystis in North America

1. Plants colonial from horizontal rhizomes; perigynia ascending ...... C. hostiana 1. Plants cespitose; perigynia (in fruit) spreading or reflexed ...... 2 2. Larger perigynia (1.8–)2–3 mm long, horizontally spreading; the beaks straight or only slightly deflexed (< 20), about a fourth to nearly half as long as the body ...... C. viridula (ssp. viridula and oedocarpa) 2. Larger perigynia 3–6.3(–6.7) mm long; beaks clearly deflexed (> 20) at least on lower portion of spike, the beak nearly or fully half as long as the body ...... 3 3. Pistillate scales yellowish green, similar in color to perigynia and inconspicuous in the spikes; widest leaves to 3.8 mm wide ...... 4 4. Larger pistillate spikes 10.5–13.5(–14) mm wide (measured beak tip to beak tip); longer perigynium beaks 2.3–3.2 mm long ...... C. viridistellata 4. Larger pistillate spikes 7–10(–11 in the North Carolina endemic C. lutea) mm wide; longer perigynium beaks (1.2–)1.3–2.3 mm long ...... 5 5. Tallest culms 25–50 cm; proximal pistillate spike bracts 1.5–4 times as long as inflorescences; peduncles of staminate spikes 0.2–0.5 length of staminate spikes; perigynium beak smooth; achenes 1–1.2 mm wide ...... C. cryptolepis 5. Tallest culms 65–125 cm; proximal pistillate spike bracts 0.5–1.3(–1.9) times as long as inflorescences; peduncles of staminate spikes mostly 0.7–2.5 length of staminate spikes; perigynium beak usually sparsely scabrous; achenes 1.2–1.5 mm wide ...... C. lutea 3. Pistillate scales strongly flushed with brown or reddish-brown, contrasting sharply with the yellowish green perigynia and hence conspicuous in the spike; widest leaves to 5.4(–5.8) mm wide ...... 6 6. Staminate spikes sessile or short-peduncled, peduncles usually less than 5 mm long; cauline leaves usually nearly as long as culms; perigynium beak usually more than 1.6 mm long ...... C. flava 6. Staminate spikes on peduncles usually more than 4.5 mm long; cauline leaves about half the length of the culms or less; perigynium beak usually less than 1.7 mm long ...... C. viridula (ssp. brachyrhyncha)

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