Effects of 2,4 D on the germination of megaspores and initial development of Regnellidium diphyllum Lindman (Monilophyta, Marsileaceae)

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1 Effects of 2,4 D on the germintion of megspores nd initil development of Regnellidium diphyllum Lindmn (Monilophyt, Mrsilecee) Cssnego, MBB., Droste, A. b nd Windisch, PG. c * Progrm de Pós Grdução em Biologi, Universidde do Vle do Rio dos Sinos Unisinos, Av. Unisinos, 950, CEP , São Leopoldo, RS, Brzil b Centro Universitário Feevle, RS , CEP , Novo Hmburgo, RS, Brzil, Universidde do Vle do Rio dos Sinos Unisinos, Av. Unisinos, 950, CEP , São Leopoldo, RS, Brzil c Progrm de Pós Grdução em Botânic, Universidde Federl do Rio Grnde do Sul UFRGS, Av. Bento Gonçlves, 9500, CEP , Porto Alegre, RS, Brzil *e mil: pteridos@gmil.com Received September 30, 2008 Accepted December 9, 2008 Distributed, (With 3 figures) Abstrct Regnellidium diphyllum is considered s endngered, occurring in the Stte of Rio Grnde do Sul, Brzil, nd few djoining loclities in Uruguy, Argentin nd the Stte of Snt Ctrin. It grows in wetlnds frequently ltered for griculturl ctivities. Herbicides bsed on 2,4 dichlorophenoxycetic cid (2,4 D) re widely used in these fields. The effects of 2,4 D on the germintion of megspores nd initil sporophytic development of R. diphyllum were investigted. Six concentrtions of 2,4 D (0.32; 0.64; 1.92; 4.80; 9.60 nd mg.l 1 ), nd the control (0.00 mg.l 1 ), were tested in vitro, using Meyer s medium. Cultures were mintined in growth chmber t 24 ± 1 C, under rtificil light with nominl irrdince of 110 µmol.m 2 /s nd 16 hours photoperiod. Megspore germintion ws lower t 9.60 nd mg.l 1 of 2,4 D (56 nd 48%, respectively), compred with the control (68%). Herbicide concentrtions of up to 1.92 mg.l 1 did not significntly decrese the number of sporophytes formed. At mg.l 1, no sporophytes were formed. The lengths of the primry root, primry nd secondry leves were greter t concentrtions of 0.32 nd 0.64 mg.l 1 of 2,4 D. Low concentrtions of 2,4 D do not ffect germintion rtes nd initil development of R. diphyllum in significnt wy. However, higher concentrtions (9.60 nd mg.l 1 ) ffect substntilly the germintion of the megspores nd interfere with the estblishment of the species. Keywords: heterosporous fern, germintion, herbicide, ecophysiology, conservtion. Efeitos do 2,4 D sobre germinção de megásporos e o desenvolvimento inicil de Regnellidium diphyllum Lindmn (Monilophyt, Mrsilecee) Resumo Regnellidium diphyllum é considerd meçd, ocorrendo no Estdo do Rio Grnde do Sul, Brsil, e em lgums locliddes vizinhs no Urugui, n Argentin e no Estdo de Snt Ctrin. Cresce em áres lgáveis, frequentemente lterds pr tividdes grícols. Herbicids bsedos em ácido 2,4 diclorofenoxicético (2,4 D) são lrgmente utilizdos nests plntções. Os efeitos do 2,4 D sobre germinção de megásporos e o desenvolvimento esporofítico inicil de R. diphyllum form investigdos. Seis concentrções de 2,4 D (0,32; 0,64; 1,92; 4,80; 9,60 e 19,20 mg.l 1 ), lém do controle (0,00 mg.l 1 ) form testds in vitro, utilizndo meio de Meyer. As culturs form mntids em câmr de germinção 24 ± 1 C, sob luz rtificil, com irrdiânci nominl de 110 µmol.m 2 /s e fotoperíodo de 16 hors. A germinção de megásporos foi menor em 9,60 e 19,20 mg.l 1 de 2,4 D (56 e 48%, respectivmente), comprd o controle (68%). Concentrções té 1,92 mg.l 1 não diminuírm significtivmente o número de esporófitos formdos. Em 19,20 mg.l 1, não houve formção de esporófitos. Os comprimentos d riz primári e ds folhs primári e secundári form miores em concentrções de 0,32 e 0,64 mg.l 1 de 2,4 D. Bixs concentrções de 2,4 D não fetm significntemente s txs de germinção e o desenvolvimento inicil de R. diphyllum. Entretnto, miores concentrções (9,60 e 19,20 mg.l 1 ) fetm substncilmente germinção de megásporos e interferem no estbelecimento d espécie. Plvrs chve: filicíne heterospord, germinção, herbicid, ecofisiologi, conservção.

2 Cssnego, MBB, Droste, A. nd Windisch, PG. 1. Introduction The contmintion of qutic environments by pollutnts such s hevy metls (Arvind nd Prsd, 2005; Rodgher nd Espíndol, 2008) nd herbicides (Coler et l., 2005; Luo nd Iked, 2007) is mjor problem for the conservtion of ntive niml nd plnt species. Wetlnds receive inputs of herbicides commonly used to control weeds through direct deposit from ircrft sprying, lterl drift nd runoff from fields. Required chrcteristics of modern herbicides include high effectiveness without cusing environmentl pollution or hrmful effects, high ctivity, low toxicity, high selectivity nd being non persistent (Usui, 2001). Rice is listed mong the mjor food crops. Its yield is substntilly reduced when weeds re not controlled. Of ll the commonly used herbicides from the phenoxy clss, 2,4 dichlorophenoxycetic cid (2,4 D) most effectively controls brod leved plnts when pplied post emergence. The herbicide presents uxinic ctivity nd stimultes the production of ethylene nd hydrogen cynide, cusing phytotoxicity (Usui, 2001). Ecotoxicologic investigtions hve mde it possible to evlute the sensitivity nd vulnerbility of plnt species to herbicides in qutic systems (Firchild et l., 1997; Forsyth et l., 1997; Wong, 2000; Sntos nd Bnztto, 2000; Sheffield, 2002; Aid et l., 2006; Luo nd Iked, 2007). Herbicides cn widely vry in the mode of ction nd effects cross vrious phylogenetic groups of plnts (Firchild et l., 1997), indicting the potentil use of qutic plnts s bioindictors. Plnts differ in their cpcity to trnsform nd degrde herbicides, since different types nd ctivities of enzymes work on this metbolism (Usui, 2001). In Brzil, the Ntionl Environmentl Council (CONAMA) llows mximum limit of 4.0 µg.l 1 of 2,4 D in wter. Concentrtions bove these vlues re considered toxic to qutic communities nd improper for humn use (MMA, 1986). Regnellidium diphyllum Lindmn, heterosporous fern in the Mrsilecee fmily, hs its distribution restricted to Southern Brzil nd some djoining loclities in Uruguy nd Argentin (Schultz, 1949; Alonso Pz nd Bssgod, 2002). It grows in wetlnds nd the rhizome develops in the humid soil or mud. The plnts re frequently subjected to flooding, with the lef lmins bove the wter surfce. In mny res, these wetlnds hve been trnsformed into rice fields nd pstures, so tht the plnts in these loclities re exposed to herbicides crried into the wter or incorported to the soil. Currently, the species is on the list of endngered species of the Stte of Rio Grnde do Sul (SEMA, 2008). It is importnt to understnd the effect of herbicides on the non trget orgnisms in qutic environments. Unfortuntely, little is known bout the effect of herbicides on qutic pteridophytes (Kery et l., 2000; Sntos nd Bnztto, 2000; Sheffield, 2002; Luo nd Iked, 2007) nd no studies re vilble for their effects on R. diphyllum. The effects of 2,4 D on the germintion of megspores nd initil sporophytic development of R. diphyllum were investigted. 2. Mteril nd Methods Mture sporocrps of Regnellidium diphyllum were obtined from different plnts collected in the municiplity of Triunfo (Stte of Rio Grnde do Sul, Brzil) nd kept t room temperture (bout 25 C) until the beginning of the experiment, one month lter. Voucher specimens were plced in the Herbrium Anchiet (PACA, São Leopoldo, Brzil). In flow chmber, sporocrps were wshed in tp wter, rinsed with 70% ethnol solution nd kept ten minutes in 7% sodium hypochlorite solution, then wshed in sterile distilled wter nd dried on sterile filter pper t room temperture. Fifteen sporocrps were crcked, thus liberting the spores, nd the megspores were seprted mnully from the microspores under stereo microscope. To ssure homogeneous megspore smples, the mteril from the different sporocrps ws mixed. Apogmy is norml occurrence in meggmetophytes of R. diphyllum (Mhlberg nd Bldwin, 1975), therefore only megspores were used, in order to obtin uniform cultures, thus voiding the mixture of sexul nd pogmiclly formed sporophytes. Meyer s solutions (Dyer, 1979) were prepred s culture medi with 2,4 dichlorophenoxycetic cid (2,4 D) t different concentrtions: 0.00 (control), 0.32, 0.64, 1.92, 4.80, 9.60 nd mg.l 1. The ph ws djusted to 6.2 before utoclving. Twenty five megspores were plced in ech glss vil ( cm) with 25 ml of the solution nd there were six repetitions for ech tretment. The cultures were mintined in growth chmber t 24 ± 1 C, under rtificil light with nominl irrdince of 110 µmol.m 2 /s nd 16 hours photoperiod. The lowest concentrtion of 2,4 D (0.32 mg.l 1 ) ws bsed on the commercil product DMA 806 BR (Dow AgroSciences Industril Ltd.), n queous solution contining 806 g.l 1 2,4 D. The mnufcturer recommends the ppliction of 1.2 L per hectre of cultivted rice (967.2 g.h 1 ). Considering the occsionl prtil submersion of R. diphyllum plnts in wter of up to c. 30 cm this would correspond to cover of three million litres of wter per hectre. In one litre of culture medium, the equivlent concentrtion of 2,4 D would be 0.32 mg.l 1. Higher concentrtions tested would represent herbicide pplictions in smll wter bodies, or dditionl input through other mens. After seven dys of culture, dt were collected weekly for totl of five weeks of observtion. In order to document the development of the sporophytes, one individul ws tken t rndom from ech replicte, in lminr flux chmber, t the end of the first nd lst week. The smples were photogrphed, mesured for the length of the primry root nd the length of the primry nd secondry leves, nd then fixed in 70% ethnol.

3 Regnellidium diphyllum germintion nd development in medium with 2,4-D After five weeks, the germinted megspores nd the meggmetophytes presenting sporophytes were counted. For ech tretment the totl number of leves ws counted for one individul from ech repetition, totlising six individuls per tretment. Megspores presenting t lest the initil globose green structure with crown of rhizoids were considered s germinted. Morphologicl detils of sporophyte development under different concentrtions were recorded. Megspore germintion nd sporophyte formtion rtes were trnsformed into percentges. The normlity nd homogeneity of the germintion rtes were verified using the Wilk Shpiro nd the Levene tests. The One Wy ANOVA test ws pplied to the dt tht conformed to normlity nd homogeneity. The difference between verges ws verified using the Tukey test with α = 0.05 probbility. The Kruskl Wllis test ws pplied to dt tht did not conform nd differences between verges were verified using the Dunn test with α = 0.05 probbility (Zr, 1999). Liner regression nlysis ws pplied to estimte the reltion between the 2,4 D concentrtions nd the respective number of leves formed. The nlyses were conducted using the Systt version 10.2 for Windows (2000) t the Universidde do Vle do Rio dos Sinos. 3. Results The ppliction of 2,4 D t high concentrtions (9.6 nd 19.2 mg.l 1 ) negtively influenced the germintion cpcity of R. diphyllum, while concentrtions up to 4.80 mg.l 1 did not differ sttisticlly from the control (Figure 1). A significnt negtive influence ws observed in the formtion of sporophytes in the tretments of 4.80 nd 9.60 mg.l 1 of 2,4 D (respectively 55 nd 31% of the meggmetophytes forming sporophytes). Lower concentrtions up to 1.92 mg.l 1 did not present significnt differences in reltion to the control, nd lso presented Germintion (%) b b b ,4-D concentrtions (mg.l 1 ) Figure 1. Germinbility of Regnellidium diphyllum Lindmn megspores in different 2,4-D concentrtions fter five weeks in culture (ANOVA test, F = 7.75, d.f. = 6, 35, P = 0.001). (F = F vlue; d.f = degree of freedom; P = probbility). Mens with different letters re significntly different by the Tukey test, α = Error brs indicte stndrd error. bc c the highest verges in sporophyte formtion. No sporophytes were formed t mg.l 1, the highest concentrtion tested (Figure 2). The length of the primry root of the sporophytes ws ffected by 2,4 D (Tble 1). After one week in culture, significnt reduction in root growth compred to the control ws observed t the concentrtion of 9.60 mg.l 1. After five weeks, these differences could still be observed in concentrtions of 1.92 mg.l 1 nd higher. The development of the primry lef ws lso ffected by the presence of 2,4 D (Tble 1). At the end of the first week, the growth of the primry lef ws significntly reduced when compred with the control t 2,4 D concentrtions of 4.80 mg.l 1 nd higher. After five weeks, the primry lef ws significntly shorter t 1.92 mg.l 1 nd higher concentrtions. The development of the first secondry lef ws strongly ffected by 2,4 D fter one week nd five weeks in culture (Tble 1). Usully it is formed fter one week in culture (Vinn, 1973). However, in the tretments with concentrtions of 4.80 mg.l 1 of 2,4 D nd higher, the secondry lef ws not formed t ll in the first week. At the highest concentrtion under which sporophyte formtion occurred (9.60 mg.l 1 ), no secondry leves developed even fter five weeks in culture. There ws significnt negtive reltion between the 2,4 D concentrtions nd the number of leves formed fter five weeks, s n observble liner regression (Figure 3). In 0.32 mg.l 1 of the herbicide, the sporophytes formed up to five leves, wheres concentrtions of 0.64 mg.l 1 nd higher cused grdul decrese of the mximum number of leves formed, declining from three to one. The men number of leves formed in ech tretment ws situted ner to the regression line. No leves were formed in the highest 2,4 D concentrtion tested. All other tretments with the herbicide presented smller numbers of leves thn the control. Gmetophytes with sporophytes (%) ,4-D concentrtions (mg.l 1 ) Figure 2. Regnellidium diphyllum Lindmn sporophyte formtion in different 2,4-D concentrtions fter five weeks in culture (Kruskl-Wllis test, H = 33.99, d.f. = 6, 35, P = 0.001). (H = H vlue; d.f = degree of freedom; P = probbility). Mens with different letters re significntly different by the Dunn test, α = Error brs indicte stndrd error. b c d

4 Cssnego, MBB, Droste, A. nd Windisch, PG. Tble 1. Regnellidium diphyllum Lindmn growth of primry root, primry lef nd first secondry lef fter one nd five weeks of in vitro culture t different 2,4 D concentrtions. Men (LB, UB) = Men vlues (lower bound, upper bound of 95% confidence intervl). Different letters in the column indicte significnt differences mong tretments by the Tukey test (α = 0.05). (F = F vlue; d.f. = degrees of freedom; P = probbility). 2,4 D (mg.l 1 ) Primry root (mm) men (LB, UB) Primry lef (mm) men (LB, UB) First secondry lef (mm) men (LB, UB) One week Five weeks One week Five weeks One week Five weeks 0.00 (control) 2.7 (1.6, 3.7) b 6.5 (5.4, 7.6) 5.3 (4.1, 6.6) 11.5 (9.9, 13.1) b 1.2 (0.6, 1.7) b 19.2 (15.7, 22.6) b (1.6, 4.7) 5.5 (4.4, 6.6) 4.7 (3.6, 5.7) 15.0 (12.6, 17.4) 1.7 (1.3, 2.1) 25.8 (18.9, 32.8) (1.9, 4.4) 6.2 (5.4, 7.0) 4.0 (2.5, 5.5) 15.5 (13.1, 17.9) 0.9 (0.1, 1.6) b 24.2 (22.1, 26.2) b (1.4, 3.6) b 3.8 ( 3.0, 4.6) b 3.7 (2.4, 4.9) b 5.8 (4.1, 7.5) c 0.6 (0.4, 0.9) bc 9.2 (4.7, 13.6) c (0.7, 1.9) bc 2.3 ( 1.2, 3.4) bc 1.8 (1.0, 2.6) bc 4.7 (2.0, 7.3) cd 0.00 c 1.9 (0.8, 4.6) d (0.4, 1.0) c 0.9 (0.2, 0.3) c 0.6 (0.5, 0.7) c 1.5 (0.6, 2.4) d 0.00 c 0.00 d F = F = F = F = F = F = d.f.= 5, 30 d.f. = 5, 30 d.f. = 5, 30 d.f = 5, 30 d.f. = 5, 30 d.f. = 5, 30 P < P < P < P < P < P < 0.001

5 Regnellidium diphyllum germintion nd development in medium with 2,4-D Number of leves Discussion ,4-D concentrtions (mg.l 1 ) Figure 3. Reltion between the number of leves of Regnellidium diphyllum Lindmn sporophytes nd different 2,4-D concentrtions fter five weeks of culture (r 2 = 0.54, F = 47.05, d.f. = 1, 40, P < 0.001). (r 2 = squred multiple r vlue; d.f = degree of freedom; P = probbiity). Blck circles represent one or more of the six replictes; white squres represent the men number of leves in ech tretment. The percentge of germinted megspores in the culture medium with 0.32 mg.l 1 of 2,4 D ws 68.5%, similr percentge of the control, which presented 68% of germintion. From 0.64 mg.l 1 onwrds, the increse of 2,4 D concentrtion ws ccompnied by the grdul decrese of germintion percentge, lthough up to 4.80 mg.l 1, no sttisticlly significnt differences were observed when compred with the control group. In the highest 2,4 D concentrtion tested (19.2 mg.l 1 ), less thn 50% of germintion ws obtined. The 2,4 D is n uxin simultor molecule tht is designed nd used s selective herbicide for brodleved plnts, i.e. dicots (Firchild et l., 1997). At low concentrtions, it stimultes growth of plnt cells nd on intercting with endogenous hormones like ethylene, giberellic cid, bscisic cid nd even uxins, 2,4 D cn brek seed dormncy, stimulting the germintion process (Brdy nd McCourt, 2003). On the other hnd, high concentrtions or successive pplictions of this herbicide retrded or even inhibited the germintion of dicotyledonous nd monocotyledonous seeds (Usui, 2001). After three dys of megspore culture, the first sporophytes could be observed. Vinn (1973) verified tht the first sporophytic structures emerged from the meggmetophytes of Regnellidium diphyllum round the first week of in vitro culture. In the present experiment, no sporophytes were obtined t mg.l 1 of 2,4 D, nd t 9.60 mg.l 1 only 31.4% of the meggmetophytes formed sporophytes. Furthermore, 4.80 mg.l 1 nd higher concentrtions cused bnorml structures. Sporophytes obtined from the tretments without 2,4 D (control) nd with the lowest 2,4 D concentrtion (0.32 mg.l 1 ) formed the lrgest number of leves (respectively, six nd five leves) fter five weeks of in vitro culture. The grdul decrese of the number of leves formed on the sporophytes, the bnorml tissue prolifertion nd the chlorosis nd necrosis of lef nd root tissues were relted to higher concentrtions of 2,4 D, specilly 4.80 to 9.60 mg.l 1. The length of the primry nd secondry leves of R. diphyllum in the tretments with minor 2,4 D concentrtion (0.32 mg.l 1 ) ws significntly higher when compred with the control group t the end of the experiment. Nevertheless, concentrtions from 1.92 to 9.60 mg.l 1 cused minor root nd primry lef growth, however no secondry leves were grown on 9.60 mg.l 1. As systemic herbicide, t high concentrtions, 2,4 D trnsloctes throughout the whole plnt, cusing biochemicl disturbnce in cells nd disorgnised synthesis of nucleic cids nd proteins (Grossmnn, 2003). Studies hve nlysed the susceptibility of ferns to rice herbicides (Sheffield, 2002; Aid et l., 2006; Luo nd Iked, 2007). However, no tests were performed on the toxicity of 2,4 D or other herbicides on R. diphyllum, so tht no direct comprisons re possible. Wong (2000) tested the concentrtions of 0.02 to 200 mg.l 1 of the herbicides 2,4 D, glyphoste nd prqut on the growth nd chlorophyll synthesis of the green lge Scenedesmus qudricud (Turpin) Brébisson. The uthor verified tht low concentrtions of 2,4 D (0.02 nd 0.2 mg.l 1 ) stimulted the two tested vribles, while 200 mg.l 1 of this herbicide inhibited completely the lgl growth nd chlorophyll production. Forsyth et l. (1997) found tht concentrtion of 0.01 mg.l 1 of 2,4 D stimulted the vegettive growth while 0.1 mg.l 1 reduced the size to 40% of the qutic mcrophytes Potmogeton pectintus L. nd Myriophyllum sibiricum Kom. In nother experiment, Sntos nd Bnztto (2000) observed tht 6.25, 12.5 nd 25 mg.l 1 of 2,4 D hd no toxic effects on two other qutic mcrophytes, Spirodel punctt (G. Mey.) C.H. Thomps. nd Slvini minim Bker, nd only Spirodel punctt ws sensitive to the concentrtion of 50 mg.l 1. Species of the qutic fern Slvini, minly S. minim nd S. molest, re frequently found in environments lso preferred by R. diphyllum. According to Sntos nd Bnztto (2000), S. minim showed tolernce to 2,4 D up to 50 mg.l 1, while in the present study the concentrtions of 4.80 mg.l 1 nd higher were toxic to R. diphyllum, showing tht this fern is more susceptible to this herbicide thn S. minim. This could be n obstcle to the estblishment nd permnence of R. diphyllum in hbitts exposed to 2,4 D contmintion. Altertions of nturl hbitt conditions, even in preserved res, the destruction of wetlnds due to expnsion of urbn res nd the estblishment of pstures nd plnttions where 2,4 D or other chemicls re used, re mjor problems to be considered in reltion to the conservtion of the currently vulnerble R. diphyllum. Acknowledgements The uthors re grteful to the Universidde do Vle do Rio dos Sinos for the use of lbortories nd the scholrship to the first uthor, to the Centro Universitário Feevle, to the Universidde Federl do Rio Grnde do Sul nd

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