Isoetes mississippiensis: A new quillwort from Mississippi, USA

Abstract Isoetes mississippiensis S.W. Leonard, W.C. Taylor, L.J. Musselman and R.D. Bray (Isoetaceae, Lycopodiophyta) is a new species known from two sites along tributaries of the Pearl River in southern Mississippi. This species is distinguished from other species in the southeastern United States by a combination of character states including a basic diploid (2n=22) chromosome count, laevigate megaspores, and a narrow velum covering less than one-third of the adaxial sporangium wall.


Introduction
Isoetes (Isoetaceae) is a cosmopolitan genus of heterosporous lycophytes containing 200-300 species (Hickey et al. 2003;Troia et al. 2016). Lycophytes have an extensive fossil record dating from the Devonian and a morphology so conserved that members of the genus Isoetes are recognized in the Triassic (Pigg 2001). Extant species are widely distributed from the tropics to the sub-arctic (Troia et al. 2016). Th ey range in habitat from evergreen aquatics to seasonal terrestrials. Resembling a tuft of chives or grass, they are easily overlooked in the fi eld.
In spite of their antiquity, widespread distribution, and diverse ecological adaptations, Isoetes species are remarkably uniform in their morphology. Plants appear simple in form with a lobed subterranean rootstock producing a tuft of linear sporophylls above and below roots along a groove between the lobes. Th is apparent morphological simplicity makes it easy to recognize a member of the genus, but diffi cult to distinguish species. Earlier taxonomists relied primarily on habitat, megaspore texture, and megaspore size to separate taxa (Engelmann 1882;Pfeiff er 1922;Reed 1965;Boom 1982). More recently, chromosome counts and molecular markers have been used to further defi ne taxa and infer their phylogeny (Taylor et al. 1993, Hoot et al. 2004Heafner and Bray 2005;Rosenthal et al. 2014).
Ornamentation and size of megaspores and microspores are important morphological features used to identify species of Isoetes. Pfeiff er (1922) erected four sections based on the megaspore ornamentation types cristate, echinate, reticulate, and tuberculate. While these sections are no longer recognized as having phylogenetic value in the genus, the emphasis on macro-ornamentation for identifi cation remains (Brunton 2015). Several categories for megaspores (cristate, echinate, laevigate, psilate, reticulate, rugulate, and tuberculate) and microspores (aculeate, cristate, echinate, laevigate, and psilate) are accepted, though there can be gradation between categories (Taylor et al. 1993;Musselman 2002). Micro-ornamentation of megaspores and microspores is sometimes recognized, but has not been included in any recent taxonomic treatments of the genus (Reed 1965;Boom 1982;Taylor et al. 1993;Brunton 2015). Generally, megaspore size increases with ploidy level (Pereira et al. 2015;Brunton 2015).
Th e habitat of species of Isoetes can be fairly specifi c and is often used in taxonomic treatments (Engelmann 1882;Reed 1965;Taylor et al. 1993;Brunton 2015). Species are generally segregated as aquatic, amphibious, or terrestrial, based on the proportion of their growing season spent in water (Engelmann 1882;Taylor et al. 1993). Some species occur only in certain habitats, such as rock pools, calcareous glades, oligotrophic lakes, and swamp forests. Widespread species such as I. melanopoda Gay and Durieu (s.s.) and I. engelmannii Braun have more varied habitat preference (Taylor et al. 1993;Brunton 2015).
Characteristics of sporophylls and rootstocks of Isoetes may also provide taxonomic information, though the utility of some of these features is questionable. Velum coverage of the sporangium, sporangium shape, sporangium wall coloration, and sporophyll length, number, color, and shape are sometimes used for species identifi cation, but these character states can be subtle and it is unclear how they may be infl uenced by environmental conditions (Engelmann 1882, Pfeiff er 1922Reed 1965, Boom 1982, Taylor et al. 1993, Brunton 2015. Cultivated plants often appear diff erent than those in situ, and spore development, photosynthetic pathways, and gene expression are signifi cantly altered by water conditions (Brunton 2015;Yang and Liu 2015;Yang and Liu 2016). However, the gestalt formed from the combination of these characters usually leads experts to accurate fi eld identifi cation.
While searching for populations of Isoetes louisianensis in southwestern Pearl River Co., MS, in the spring of 1996, one of us (Leonard) discovered a population of Isoetes that did not appear to be I. louisianensis or any other known species. Th ese plants had very long and numerous sporophylls bearing megaspores with a smooth surface rather than an irregularly reticulate texture that is typical of I. louisianensis megaspores. In addition, the megaspores of this plant were noticeably smaller than those of I. louisianensis. Further investigation turned up a second population downstream in Lotts Creek. Both of these waterways are tributaries of the Pearl River, converging near Picayune, MS.

Methods
Field work was performed in 1996, 1998, and 2013 to obtain specimens for further study. Specimens were deposited in the Old Dominion University herbarium (ODU). Length and width of the rootstock, sporophylls, and sporangia were measured for 10 individuals. Megaspores and microspores were removed from sporangia, cleaned by sonication in distilled water for 90 seconds, and dried on a slide warmer at maximum temperature (approximately 60°C). Light images were captured using a Nikon SMZ800 stereomicroscope with attached Digital Sight camera, and measurements made within the Digital Sight control panel. Spores were prepared for scanning electron microscopy by coating with 25 nm of gold-palladium using a Cressington high resolution sputter coater (Cressington Scientifi c Instruments Ltd.). Imaging was performed on a Zeiss EVO MA 15 scanning electron microscope. Chromosome counts were determined by root tip squashing as described in Heafner and Bray (2005). Site descriptions were prepared and lists of associated species were made.

Results
Analysis of morphological characters, chromosome counts, and ecological evaluation leads us to conclude our collections represent an undescribed species of Isoetes. Description. Plants amphibious in and along persistent streams. Rootstock subglobose, bilobed, brown, 0.5−1.0 cm long, 1.0−1.5 cm wide. Roots dichotomously branched. Sporophylls (leaves) linear, bright green, darkening with age, pale toward base, spirally arranged, erect to spreading, up to 40 cm long and 2.0 mm wide at midlength, in tufts of ca. 20, semi−terete with adaxial surface fl attened, becoming more terete distally, with translucent alae ca. 1 mm wide extending along lateral edges from base to ca. one-quarter leaf length, tapering gradually toward apex, abruptly dilated and spatulate toward base where streaks of brown pigmented cells are often evident on pale outer surface of leaf base. Ligule triangular, ca. 1 mm long. Sporangium ovate, most 4−10 mm long, most 4−5 mm wide, adaxial wall spotted to streaked with scattered clusters of brown pigmented cells. Velum incomplete, covering less than one third of sporangium wall. Megaspores globose, white, trilete, macro-ornamentation laevigate with echinate micro-ornamentation, ca. 280−380 μm in diameter, averaging ca. 340 μm. Microspores broadly fusiform, macro-ornamentation echinate with bacillate micro-ornamentation, pale brown in mass, monolete, 25−30 μm long.

Cytology. Chromosome counts show individuals of Isoetes mississippiensis to be diploid (2n=22).
Ecology. Isoetes mississippiensis occurs in sluggish, persistent streams in southern Mississippi (Figure 4). At the Moody Branch locality, the maintained right-of-way of Mississippi Highway 43 allows abundant sunshine to reach the stream and adjacent wetlands. Small bushes and saplings of titi (Cyrilla racemifl ora) and red maple (Acer rubrum) are periodically cut down and allowed to fall in the stream. Sediment and detritus provide anchors for herbaceous growth of sedges, rushes, and coarse grasses (Rhynchospora inexpansa, Juncus spp., Erianthus giganteus, Panicum spp.). In the shallow water stream margin is Iris virginica. Th e woodland edge is suitable habitat for crossvine (Bignonia capreolata) and rattan vine (Berchemia scandens). Upstream where a defi ned channel is present the overstory consists of swamp black gum (Nyssa bifl ora), laurel oak (Quercus laurifolia), red maple, and encroaching loblolly pines (Pinus taeda). Shrubs in the understory are Elliott's blueberry (Vaccinium elliottii), yaupon (Ilex vomitoria), and titi. In the upper reaches of Moody Branch, the channel is braided and the water sluggish, more typical of a swamp black gum forest with Rankin's jessamine (Gelsemium rankinii), Virginia willow (Itea virginica), and dog hobble (Viburnum nudum).
After fl owing west for several kilometers, Moody Branch turns sharply south just west of Mississippi Highway 43 and eventually merges with Lotts Creek. Th e forested wetland adds pond cypress (Taxodium ascendens) and a dense shrub understory with Smilax laurifolia. At the Walkiah Bluff Road crossing of Lotts Creek disturbance has been severe, yet I. mississippiensis has revegetated new habitat in the roadside ditch north of the road and on sandbars.

Discussion
Evaluation of the morphological and cytological features of I. mississippiensis shows it to be distinct from all other taxa in the southeastern US. In the coastal plain of the Gulf Coast states, nine other species are known: I. appalachiana, I. boomii, I. fl accida s.l., I. hyemalis, I. louisianensis, I. melanopoda s.l., I. microvela, I. texana, and I. valida (Singhurst et al. 2011;Weakley 2015;Brunton 2015). A basic diploid chromosome count (2n=22) plus laevigate megaspore ornamentation separates I. mississippiensis from all these taxa except I. texana and occasionally I. melanopoda. Th ese species may be further separated by presence/absence of phyllopodia, diff erence in megaspore size, and velum coverage (Table 1). Additionally, the habitats of these species are quite diff erent. Isoetes mississippiensis occurs along persistent streams, while I. texana is found in freshwater ponds and interdunal swales and I. melanopoda grows in wet prairies, soil pockets on rock outcrops, and woodland depressions (Table 1; Singhurst et al. 2011;Taylor et al. 1993