J. Aquat. Manage. 49: 122-125 Vegetative propagation in the emergent macrophyte American water willow ( americana L. Vahl)

BRANT TOUCHETTE, MARIANA POOLE, MICA MCCULLOUGH, EMILY ADAMS, AND CARL NIEDZIELA*

INTRODUCTION 2005). Nevertheless, this concern is offset by the advantages water willow provides in terms of enhancing streambed and Over the past few decades, numerous attempts have been shoreline stability and modifying habitat to support associat- made to establish aquatic vegetation within littoral zones of ed organisms (Keiper et al. 1998, Fritz et al. 2004b). In hy- freshwater lakes and reservoirs as a means of enhancing the drologically dynamic systems, water willow has been shown to productivity of recreationally important fishes, improving wa- tolerate scouring floods, intense wave action, and fluctuating ter quality, and/or minimizing shoreline erosion (e.g., Qui water levels (Penfound 1940, Lewis 1980, Fritz and Feminella et al. 2001, Strakosh et al. 2005). The success of these plant- 2003, Strakosh et al. 2005). Therefore, in barren littoral ar- ings is often dependent on grazing intensities from herbi- eas, with comparatively intense hydrologies (e.g., high water vores that remove important aboveground structures or velocities or varying water levels), water willow may be a de- uproot vegetation through benthic feeding, and the ability sirable candidate for establishing emergent vegetation. The of to survive dynamic hydrologies (Nishihiro et al. success of water willow in these areas, however, may be de- 2004, Strakosh et al. 2005, Touchette et al. 2007). One prom- pendent on its size, health, and maturity at the time of plant- inent North American plant that seems to be somewhat toler- ing. In regions where natural populations of water willow are ant of these biotic and abiotic perturbations is American limited and/or protected, greenhouse propagation could al- water willow, Justicia americana L. (Vahl.) (Lewis 1980, Hill low for the mass production of a large number of planting 1981, Strakosh et al. 2005, Touchette and Frank 2009). This units from only a few cuttings. tolerance is likely fostered by its complex network of below- The purpose of this study was to experimentally evaluate ground structures and its remarkable ability to regenerate culture techniques for rapid greenhouse propagation of wa- new aboveground tissues following major disturbances (Fritz ter willow from stem cuttings. More specifically, we com- et al. 2004a). pared how flooded and water-saturated conditions The native range of water willow includes the eastern half influenced new shoot production and growth, and evaluated of North America from Quebec to Florida and westward into the use of a root-promoting hormone for enhancing root Texas (Niering and Olmsted 1979). In some parts of the Mid- growth. western United States, water willow is either threatened (Michigan) or endangered (Iowa) and is classified as a spe- cies of concern in Canada. In North Carolina, it is consid- MATERIALS AND METHODS ered a principle habitat species for the endangered (Notropis mekistocholas; Hewitt et al. 2009), and al- In early July 2009, approximately 150 cuttings were col- though restoration programs involving water willow along lected from a midmarsh region of Badin Lake (a reservoir the middle Cape Fear River have yet to be initiated, it is likely near Albemarle, NC). Plants were cleaned and processed by to be a necessary component in maintaining viable Cape removing chlorotic or necrotic tissue, and stems were cut Fear shiner populations in the future. to length (approximately 45 cm). The initial numbers of In shallow lake and river systems (<1.5 m), water willow is leaves and nodes per cutting were 12 to 22 and 7 to 9, respec- among the first colonizers of barren substratum where it of- tively. Six water willow cuttings were equally selected from ten stabilizes the shoreline with its massive network of below- harvested stock and placed in each tray (standard 1020 flats) ground structures (Lewis 1980, Strakosh et al. 2005). This containing 2 L of sand. A total of 20 trays were placed in a colonial plant quickly spreads along shorelines, often form- randomized complete block design within a glass research ing highly dense monocultures (Lewis 1980, Hill 1981). Be- greenhouse, with 4 different treatments composed of (a) wet cause of its aggressive rhizomatous growth, in some regions sand: moist well-drained sand (a widely used propagation water willow is considered a pest species (Strakosh et al. substrate for aquatic plants; Baca and Ballou 1989, Schaff et al. 2003); (b) flooded sand: water levels maintained at 4 to 5 cm above the sand substrate, (c) wet sand + hormone: well- *First, third, and fourth authors: Associate Professor and two students, drained sand with root-promoting hormone; and (d) flood- Center for Environmental Studies, Elon University, Elon, NC 27244; second ed sand + hormone: flooded trays with sand and root-pro- and fifth authors: student and Assistant Professor, Department of Biology, Elon University, Elon, NC 27244. Corresponding author’s E-mail: moting hormone (n = 5 trays for each treatment). The root- [email protected]. Received for publication January 3, 2011 and in promoting hormone was a readily available product (Root- revised form March 23, 2011. ing Hormone, Green Light Co., San Antonio, TX) common

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Figure 1. American water willow shoot and root characteristics including (A) new shoots per cutting, (B) new shoots per node, (C) total new shoot mass, (D) number of nodes with roots, (E) total root mass, and (F) number of shoots with roots. Data include wet sand without hormone (white bars), wet sand with hormone (gray bars), flooded sand without hormone (dark gray bars), and flooded sand with hormone (black bars). Data are presented as means ± 1 SE. Means with different letters are significantly different according to Tukey-Kramer post-hoc analyses (α = 0.05).

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in many horticultural supply stores, and contained 0.1% of Unlike results for shoots, the number of nodes that devel- the active phytohormone indole-3-butyric acid (a synthetic oped roots was affected by both flooding and root-promot- auxin similar in structure to indole-3-acetic acid). The hor- ing hormone. In this case, there was a slight, albeit mone was applied to each node according to the manufac- significant, positive response with hormone applications (p = turer’s instructions. To prevent nutrient growth limitation, 4 0.022; Figure 1d) which was most pronounced in the second g of a slow release fertilizer (Osmocote 14-14-14) was added month (Aug). This difference, however, was no longer evi- to each tray at the time of planting. Throughout the study, dent by September. Greater root mass was observed in flood- trays with cuttings were watered for 10 min, 3 times a day ed plants (3.10 ± 0.36 g of new root tissue per cutting) than (2:00 AM, 10:00 AM, and 2:00 PM) with overhead misters. In- plants in wet sand (1.21 ± 0.29) at the end of the study (p < dividual watering delivered approximately 0.5 cm of water to 0.0001; Figure 1e). There were no significant differences in each tray, totaling 1.5 cm of water d-1. At this rate, wet-sand the number of new shoots with roots between any treat- treatments remained moist throughout the study. ments. Root development seemed to lag behind shoot devel- At monthly intervals, a single cutting was removed from opment, and the onset of root growth was likely contingent each tray and measured for new shoot production; measure- on the initiation of shoots. ments included number of shoots per cutting, number of Based on the results of this study, we conclude that water shoots per node, and total new shoot mass. We also consid- willow is readily propagated from stem cuttings, which is con- ered root development by measuring total root mass, num- sistent with observations made in natural settings (Lewis ber of nodes with developing roots, and number of new 1980). Most cuttings produced new shoots, including those shoots with roots. Mass of plant tissue was determined by ov- that developed mild necrosis in leaf and stem tissue. Shoot en drying at 60 C until constant weight. and root mass was significantly higher for plants cultured in All growth parameters were evaluated for equal variance flooded sand than for plants in wet sand. While we observed and normality (Kolmogorov-Smirnov test). Shoot and root a significant response in shoot and root development follow- weights were log transformed, and the number of shoots per ing some hormone applications, we believe that these bene- cutting was square-root transformed to satisfy normality prior fits were not substantial and could not justify the added to statistical comparisons. Data were analyzed using a 3-way expense and time necessary to apply root-promoting hor- repeated measures ANOVA (GLM; SAS 9.1) with measured mone to water willow nodes. shoot and root parameters as dependent variables, and hor- mone, water conditions, and time as independent variables. ACKNOWLEDGEMENTS A Tukey-Kramer post-hoc analysis was performed when sig- nificant differences were reported (α = 0.05). This study was partially supported by the Water Resources Research Institute of the University of North Carolina, the US Geological Survey, North Carolina Sea Grant, and the RESULTS AND DISCUSSION Elon University’s Center for Environmental Studies. We Regardless of treatment, water willow cuttings were ca- thank two anonymous referees for their helpful comments pable of producing new vegetative shoots. After 3 months, and constructive suggestions on an earlier version of this the mean number of new shoots per cutting was between manuscript. 6.2 and 11.4; plants in the wet sand + hormone treatment had the highest numbers (Figure 1a). Most of the shoots LITERATURE CITED emerged during the first month, and subsequent increases Baca BJ, Ballou TG. 1989. Propagation of woody wetland vegetation for in- in plant mass were primarily due to increases in shoot size kind mitigation, pp. 1-9. In: F.J. Webb (ed.). Proceedings of the 16th and/or continued apical growth of the original cutting Annual Conference on Wetland Restoration and Creation. Hillsborough (data not presented). There was a significant shoot re- Community College, Institute for Florida Studies, Tampa, FL. sponse associated with water treatments, wherein flooded Fritz KM, Evans MA, Feminella JW. 2004a. Factors affecting biomass alloca- treatments had less shoot proliferation by the end of the tion in the riverine macrophyte Justicia americana. Aquat. Bot. 78:279-288. Fritz KM, Feminella JW. 2003. Substratum stability associated with the river- study (p = 0.030; Figure 1a). While statistically significant, ine macrophyte Justicia americana. Freshwater Biol. 48:1630-1639. these outcomes are mostly attributed to the markedly Fritz KM, Gangloff MM, Feminella JW. 2004b. Habitat modification by the higher performance observed in the wet + hormone treat- stream macrophyte Justicia americana and its effects on biota. Oecologia. ment rather than the overall performance of both wet- 140:388-397. Hewitt A, Kwak TJ, Cope WG, Pollock KH. 2009. Population density and in sand treatments collectively (Figure 1a). Nevertheless, this stream habitat suitability of the endangered Cape Fear shiner. T. Am. response is consistent with field observations, where new Fish. Soc. 138:1439-1457. shoot production was reduced by 28% when growing in 2 Hill BH. 1981. Distribution and production of Justicia americana in the New cm of water in comparison to plants lying directly on the River, Virginia. Castanea. 46:162-169. substratum (Lewis 1980). Shoot mass was significantly Keiper JB, Brutsche PL, Foote BA. 1998. Acalyptrate diptera associated with water willow, Justicia americana (). Proc. Entomol. Soc. Wash. greater in flooded treatments than in wet-sand treatments 100:576-587. at the end of the study (p < 0.0001; Figure 1c). Shoots Lewis K. 1980. Vegetative reproduction in populations of Justicia americana in were more than 3 times larger in flooded sand (3.72 ± 0.65 Ohio and Alabama. Ohio J. Sci. 80:134-137. [SE] g of new shoot per cutting) than in wet sand (1.04 ± Niering WA, Olmstead NC. 1979. National Audubon Society field guide to North American wildflowers, eastern region. Knopf, NY. 887 pp. 0.28 g). This suggests that growth in flooded conditions Nishihiro J, Miyawaki S, Fujiwara N, Washitani I. 2004. Regeneration failure may enhance overall productivity, resulting in larger and of lakeshore plants under an artificially altered water regime. Ecol. Res. presumably healthier plants. 19:613-623.

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Penfound WT. 1940. The biology of Dianthera americana L. Am. Midl. Nat. Touchette BW, Frank A. 2009. Xylem potential- and water content-break- 24:242-247. points in two wetland forbs: indicators of drought resistance in emergent Qui D, Wu Z, Liu B, Deng J, Fu G, He F. 2001. The restoration of aquatic mac- hydrophytes. Aquat. Biol. 6:67-75. rophytes for improving water quality in a hypertrophic shallow lake in Touchette BW, Iannacone LR, Turner G, Frank A. 2007. Drought tolerance Hubei Province, China. Ecol. Eng. 18:147-156. versus drought avoidance: A comparison of plant-water relations in her- Schaff SD, Pezeshki SR, Shields FD. 2003. Effects of soil conditions on sur- baceous wetland plants subjected to water withdrawal and repletion. Wet- vival and growth of black willow cuttings. Environ. Manage. 31:748-763. lands. 27:656-667. Strakosh TR, Eitzmann JL, Gido KB, Guy CS. 2005. The response of water wil- low Justicia americana to different water inundation and desiccation regimes. N. Am. J. Fish. Manage. 25:1476-1485.

J. Aquat. Plant Manage. 49: 125-128 Floristic account of submersed aquatic angiosperms of Dera Ismail Khan District, northwestern Pakistan

SARFARAZ KHAN MARWAT, MIR AJAB KHAN, MUSHTAQ AHMAD AND MUHAMMAD ZAFAR*

INTRODUCTION and root (Lancar and Krake 2002). The aquatic plants are of various types, some emergent and rooted on the bottom and Pakistan is a developing country of South Asia covering an others submerged. Still others are free-floating, and some area of 87.98 million ha (217 million ac), located 23-37°N 61- are rooted on the bank of the impoundments, adopting 76°E, with diverse geological and climatic environments. The semiaquatic habitat (Ahmad and Younis 1979). annual rainfall ranges from 12.5 cm (4.92 in) in the south to In Pakistan, the weediest aquatic species belong to the 87.5 cm (34.5 in) in the submountainous and the northern submerged group that germinates, sprout, grow, and repro- plains. About 70% of the rain falls during the monsoon sea- duces beneath the water surface. Their roots and reproduc- son (Jul-Sep); however, occasional showers also occur during tive organs remain in the soil at the bottom of the water the winter. The summer months are very hot, except in the body. These species cause the most damage because, de- mountainous areas while the winter months are mild in the pending on the degree of their intensity and growth, they plains and severe in the mountains (Ahmad et al. 2007). are not visible on the surface and they impede the flow of wa- Dera Ismail Khan is located in northwestern Pakistan and ter (Lancar and Krake 2002) causing overflows that lead to has an elevation of 173 m. It has a total geographical land loss of irrigation water (Iqbal 1992). Most of these species are mass of 0.896 million ha (2.214 million ac) of which 33% is found in shallow and medium deep water bodies and in flow- cultivated (Khan 2003). The climate is continental with ing canals and drainage ditches (Lancar and Krake 2002). marked temperature fluctuations both seasonal and diurnal, Some, such as Hydrilla verticillata, Potamogeton crispus and P. with significant aridity. January is the coldest month of the pectinatus, increase sedimentation of water reservoirs at accel- year and July the hottest. The mean maximum and mini- erated rates (Ashiq et al. 2003). Nevertheless, submerged mum temperatures during winter are 20.3 C and 4.2 C, re- vegetation is mostly associated with a healthy aquatic system spectively, compared to 42 C and 27 C during summer. because the plants provide habitat, sediment stabilization, Average annual rainfall is 259 mm (Chaudhry 1999). primary production, and sources of food for many birds, Worldwide there are more than 100 families of vascular mammals, fish, and insects (Ahmad and Younis 1979). They aquatic plants. These plants are structurally different from are a source of oxygen for respiration and provide protec- mesophytes or xerophytes by having less developed protec- tion through temperature moderation against hot and cold tive and conductive tissues. They have unique adaptations weather. for buoyancy and aeration, particularly in the ground tissue Lakes and ponds are rich in aquatic flora that constitute of the petiole and leaf mesophyll and in the cortex of stem an important resource but in Pakistan these natural resourc- es have not been given due attention, and thus their poten- tial remains unexplored. In addition, aquatic plants can be *First author: University Wensam College, Gomal University Dera Ismail taxonomically difficult, and Pakistan lacks adequate herbari- Khan, KPK, Pakistan. Second, third, and fourth authors: Department of um material to represent the variability in the development Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Correspond- ing author’s E-mail: [email protected]. Received for publication of various organs resulting from plasticity in form and struc- November 8, 2010 and in revised form May 31, 2011. ture in relation to aquatic environment. The peak flowering

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