the phylogenetic position of river-weeds podostemaceae insights from rbcl sequence data

8/4/2019 The Phylogenetic Position of River-weeds Podostemaceae Insights From RbcL Sequence Data

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E L S E V I E R Aquatic Botany 57 (1997) 5-2 7

Th e phylogene t ic p os i tion o f river-weeds(Podostemaceae)" Insights from r b cL sequence data

D o n a l d H . L e s a ,* , C . T h o m a s P h i lb r i c k b, A l e j a n d r o N o v e l o R . ca Department o f Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269-3042, USA

b Department of Biology, Western Connectwut State Unit,erst~, Danbury, CT 06810, USAc Departamento de Botrnica, Instituto de Biologla, Universidad Nacional Autdnoma de Mdxtco,

Postal 70-233, 04510 MJxico, D.F., MJxico

Ab s t r a c t

T h e s y s t e m a t i c p o s i t i o n o f t h e r i v e r - w e e d f a m i l y P o d o s t e m a c e a e r e m a i n s e n i g m a t i c d u e t ot a x o n o m i c d i f f i c u l t i e s i m p o s e d b y t h e r a d i c a l l y a l t e r e d m o r p h o l o g y o f t h e s e a l g a - l i k e a n -g i o s p e r m s . A l t h o u g h p r e v i o u s w o r k e r s h a v e p l a c e d t h is g r o u p p h y l o g e n e t i c a l l y a m o n g a w i d ev a r i e t y o f m o n o c o t y l e d o n s a n d d i c o t y l e d o n s , m o s t c o n t e m p o r a r y a u t h o r s h a v e p r o p o s e d t h a tf i v e r -w e e d s a r e c l o s e l y r e l a te d t o m e m b e r s o f t h e d i c o t y l e d o n o u s o r d e r R o s a l e s. A d i v e r s i ty o fop in ion a l so ex i s t s a s to whe ther the Hyc l ros t achyaceae a r e r e l a t ed to Podos tem aceae . W e hav ei n v e s t i g a t e d t h e p h y l o g e n y o f f i v e r - w e e d s b y c o m p a r i n g D N A s e q u e n c e s o f t h e c h l o r o p l a s te n c o d e d rbcL gene fo r e igh t r ive r -weed genera toge ther wi th 84 o the r ang iosperm and 11n o n - f lo w e r i n g s e e d p l a n t t a x a . T h e h i g h l e v e l o f s e q u e n c e d i v e r g e n c e i n rbcL t ha t ex i s t s be tweenf i v e r- w e e d s , H y d r o s t a c h y a c e a e a n d o t h e r a n g i o s p e r m s p r e s e n t s s y s t e m a t i c p r o b l e m s t h a t p a r a l le lt h o s e a s s o c i a t e d w i th t h e h i g h l y d i v e r g e n t m o r p h o l o g y o f t h e s e g r o u p s . R o o t i n g rbcL sequencesw i t h d i s t a n t n o n - fl o w e r i n g p la n t o u t g r o u p s r e s u l ts i n a t o p o l o g y w h e r e P o d o s t e m a c e a e c o m p r i s e ab a s a l a n g i o s p e r m c l a d e , b u t i n w h i c h o t h e r r e n d i t i o n s o f a n g i o s p e r m f a m i l y r e l a t i o n s h i p s a r ed e p i c t e d u n r e a s o n a b l y . R e s t r i c t i n g t h e c o m p a r i s o n o f r i v e r - w e e d s e q u e n c e s e n t i r e l y w i t h a n -g i o s p e r m s p l a c e s t h e g r o u p a s a s i s t e r c l a d e t o t h e H y d r o s t a c h y a c e a e a s s o m e a u t h o rs h a dan t i c ipa ted , bu t t h i s r esu l t i s on ly weak ly suppor t ed . The h igh l eve l o f bo th morpho log ica l andmolecu la r d ive rgence in the r ive r -weed c l ade confounds e f fo r t s t o co r r ec t ly asce r t a in the i rphy logene t i c r e l a t ionsh ips . A t en ta t ive hypo thes i s f rom rbcL d a t a i s t h a t t h e H y d r o s t a c h y a c e a ea n d P o d o s t e m a c e a e a r e s i s t e r t a x a w h o s e c l o s e s t r e la t iv e s a r e t h e r o s i d f a m i l i e s C r a s s u la c e a e a n dHalo ragaceae . 1997 E l sev ie r Sc ience B.V.

* C orresponding author.0304-3770/97/$17.00 1997 Elsevier Science B.V . A ll rights reserved.PH S 0 3 0 4 - 3 7 7 0 ( 9 6 ) 0 1 1 1 7 - 5


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6 D .H . Les e t a L / A q u a t i c B o t a n y 5 7 ( 19 9 7) 5 - 2 7K eyw o r d s : Ch lorop last D NA ; Cla dist ics; Crassulaceae; Evolut ion; Haloragaceae; H ydrostachyaceae; O ut-group; Parsimony; Pod ostem oide ae; Rosales; Saxifragaceae; Systematics; Tr is t ichaceae; T r is t ichoid eae

1 . I n t r o d u c t i o n

Phylog ene t i c re la t ionsh ips o f aqua t i c ang iospe rm s o f ten a re d i f f i cu l t to reconc i l ebecause adap ta t ion to hydr ic hab i t a t s typ ica l ly i s accompan ied by ex tens ive s t ruc tu ra lreduc t ion and m odi f i ca t ion tha t can obscure o r even e l im ina te t axonom ica l ly use fu lcha rac te r s . Th i s p rob lem i s exace rba ted in the ' r ive r -weeds ' (Podos temaceae ) whoselo t i c r ive r rap id hab i t a t s have s e lec ted fo r h igh ly unusua l and ex t reme s t ruc tu ra lmodi f i ca t ions . Taxonomis t s have t rea ted the o rde r Podos tema les L ind ley a s compr i s inge i the r the s ing le fam i ly Podo s temaceae R ich . ex C . A gardh (e .g . Cronqu is t , 1981 ;Steenis , 1981; Co ok, 1990) or tw o dis t inc t fam il ies , Tr is t ichaceae W il l is and Po-dos tem aceae (e .g . Wil l is , 1915, 1926; Cusse t and Cu sse t , 1988a) . The for m er (sensula to ) concep t usua l ly recogn izes two subfami l i e s , T r i s t i cho ideae and Podos temoideae(Van R oyen , 1951 ; Takh ta jan , 1980 ; Thorne , 1992). A l though m os t t axonom is t s havefo l low ed a s ensu l a to concep t o f Podos temaceae , recogn i t ion o f T r i s ti chaceae has mer i t(see discuss ion) . The Podos temaceae (s .1 . ) have a lso been cons idered as c lose re la t ivesof H ydros tach yaceae , because o f va r ious s imi la r i ti e s and the i r unusua l a f f in i ty fo rrheophy t ic hab i t a t s.

R ive r -w eed adap ta t ion has re su l ted in an un or thodo x morpho log ica l o rgan iza t ion tha ta u th o r s h a v e d e sc r ib e d a s " a l g a - l i k e " , " f u c o i d " , " l i c h e n - l i k e " , " m o s s - l i k e " o r" w e b b e d " ( G r a h a m a n d W o o d , 1 97 5; C r o n q u is t , 1 98 1; V a n S t e e n is , 1 9 81 ). A m o n g t h epecu l i a ri t ie s o f r ive r -weeds a re a ' t ha l lus ' and spec ia l i zed adhes ive ' hap te ra ' tha tfac i l it a t e the i r a tt achm ent to rock subs tra te s (Grah am and W ood , 1975 ; Cronqu is t , 1981 ;Van Steenis , 1981; J~iger-Ziirn, 1992; Rutishanser and Huber, 1995; Rutishauser, 1997).Some fea tu res o f f ive r -weeds a re so ex tens ive ly modi f i ed tha t they supe r f i c ia l lyre semble m ono co ty led on cha rac te r i st i c s. The p resence o f s i l ica bod ies , t r imerous gynoe -c ia , and fused s t amen f i l aments a re remin i scen t o f cond i t ions found in the h igh lyspec ia li zed m ono co ty led on fam i ly Orch idaceae (Dah lg ren , 1980). The P odos tem aceaeun ique ly pos ses s a ' p seud oem bryo sac ' tha t re su l ts f rom d i s in teg ra tion o f nuce l l a r ce l l sbe low the embryo sac ; the re i s a l so no t r ip le fus ion , and hence no endospe rmdeve lo pm ent in the g roup (W ent , 1910 ; Bho jw an i and Bha tnaga r , 1979). The re i sev idence tha t the p lum ule abor t s rap id ly in s eed l ings , in w hich case the tha l lo id g rowthfo rm cou ld rep resen t a h igh ly spec ia l ized , pho tosyn the t i c roo t sys tem (Dorm er , 1972 ;Fahn , 1982). Ho wev er , Ru t i shause r (1997) desc r ibed shoo t s a s rosu la te , tha l lo id o re longa te in fo rm and a r i s ing endog enou s ly f rom roo t s. Even the in te rp re ta t ion o f bas iclea f s t ruc tu re in Podo s temaceae i s com plex (Ru t i shause r , 1995 , 1997).

T h e c o m b i n a t i o n o f u n c o n v e n t i o n a l m o r p h o l o g y , u n u s u a l e m b r y o l o g y , a n d r e d u c e dana tom y has l ed to a vas t d i f fe rence o f op in ion rega rd ing the sys tem a t ic re la tionsh ips o fr ive r -weeds . As summar ized in an ea r ly t axonomic h i s to ry (Van Royen , 1951) , Po-dos tem aceae were f i r s t desc ribed (Hu m bold t e t a l. , 1816) a s a fam i ly re la ted tomonoco ty ledons (Al i sma taceae , Bu tomaceae , Juncaceae ) , a conven t ion fo l lowed bymany subsequen t au thors . La te r op in ions sugges ted a f f in i t i e s to o the r monoco ty ledons


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D .H . Le s e t a l. / A q u a t i c B o t a n y 5 7 ( 19 9 7) 5 - 2 7 7

i nc l ud i ng N a j adacea e (Schu l t z , 1832), Le m nac eae and even O rch i daceae (Pres l, 1830).O t he r unconven t i ona l a s soc i a t i ons w ere made w i t h t he f ami l i e s C era t ophy l l aceae ,Cal l i t r i chaceae , and even the a lga l Characeae (Schul tz , 1832; Endl icher , 1837, 1839) .

L i n d l e y ( 1 8 3 0 ) , S c h l e i d e n ( 1 8 3 9 ) a n d G a r d n e r ( 1 8 4 7 ) d e m o n s t r a t e d t h e d i c o t y l e d o nembryo o f Podos t emaceae . H ow ever , even w i t h t h i s i n t e rp r e t a t i on , t he a f f i n i t i e s sug-ges t ed fo r t he f ami l y ( i nc l ud i ng P i pe raceae , N epen t haceae , Po l ygonaceae , Sc rophu l a r i -a c e ae , L e n t i b u l a ri a c e a e , a n d C a r y o p h y l l a c e a e ) r e m a i n e d v a g u e ( V a n R o y e n , 1 95 1:G raham and Wood , 1975) . Even t ua l l y , embryo l og i ca l s t ud i e s f ocused a t t en t i on onR osaceae , Sax i f r agaceae and C ras su l aceae a s pos s i b l e r e l a t i ves o f Podos t emaceae(Eich ler , 1886: W an nin g, 1888; W i l l is , 1902; W ent , 1910: Ro m bac h, 1911) . Arb er( 1 9 2 0 ) r e c o u n t e d e a r l i e r o p i n i o n s t h a t P o d o s t e m a c e a e r e p r e s e n t e d a n a n c i e n t g r o u pre l a t ed t o R osa l e s and Sa r r acen i a l e s and she sugges t ed a f f i n i t i e s t o t axa such a sN e p e n t h e s and Sax i f r agaceae . Subsequen t au t hor s (H u t ch i nson , 1926 ; Eng l e r , 1930 ;Maur i t zon , 1933) con t i nued t o suppor t t he r e l a t i onsh i p o f Podos t emaceae t o t hesef ami l i e s . V an R oyen (1951) conc l uded t ha t Sax i f r agaceae w ere e s sen t i a l l y t he s i s t e rg r o u p t o a c o m p l e x c o m p r i s i n g R o s a c e a e , C r a s s u l a c e a e a n d P o d o s t e m a c e a e , w i t h t h el a t t e r t w o f ami l i e s r e l a t ed mos t c l ose l y . Scu l t ho rpe (1967 , p . 21 ) no t ed t ha t t hep h y l o g e n e t i c p o s i ti o n o f P o d o s t e m a c e a e w a s " o b s c u r e d b y t h e v e r y st ra n g e m o r p h o l o g yof a l l i t s member s " , bu t be l i eved t he r e w as su f f i c i en t ev i dence t o sugges t a r e l a t i onsh i pt o Sax i f r agaceae ( supe r i o r ova ry , f r ee s t y l e s , numerous ana t ropous ovu l es ) and C ras su -l aceae (va r i ous embryo l og i ca l f ea t u r e s ) . Takh t a j an (1980) r ega rded t he Podos t emaceaea s " r e l a t e d t o a n d d e r i v e d f r o m " t h e o r d e r S a x i f r a g a l e s , a n d f o l l o w e d s e v e r a l a u t h o r s( e .g . Mau r i tzon , 1933 ; K ap i l , 1970; M aheshw ar i , 1945) w h o conc l ud ed t ha t t he f ami l yw a s d e r i v e d f r o m C r a s s u la c e a e .T h e r o s a l e a n a l l i a n c e o f P o d o s t e m a c e a e h a s n o t b e e n a c c e p t e d w i t h o u t r e s e r v a t i o n .C ronqu i s t ( 1981) sha r ed t he op i n i on t ha t Podos t emaceae a r e r e l a t ed t o e i t he r Sax i f r a -g a c e a e o r C r a s s u l a c e a e b u t s t i l l c o n s i d e r e d t h e m t o b e " t a x o n o m i c a l l y i s o l a t e d " . V a nSt een i s ( 1981) a l so a s sumed t ha t Podos t emaceae w ere r e l a t ed t o R osa l e s , bu t empha-s i z e d t h a t f u r t h e r d e t a i l s o f t h e i r a n c e s t r y w e r e " o b s c u r e " . D a h l g r e n ( 1 9 8 0 ) p l a c e dP o d o s t e m a c e a e i n s u p e r o r d e r P o d o s t e m i f l o ra e , c o m m e n t i n g t h a t " . . . t h e y a r e s o s p e c ia l-i zed t ha t t hey canno t be a s soc i a t ed w i t h any o t he r supe ro rde r w i t hou t s eve re r e se rva -t i o n s " . C o m e r ( 1 9 7 6 ) a r g u e d t h at th e s e e d s t ru c t u re o f P o d o s t e m a c e a e w a s s o d i f fe r e n tf rom C ras su l aceae o r Sax i f r agaceae "a s t o d i s c r ed it t he i dea " o f t he i r r e l a ti onsh i p , andbe l i eved t ha t t he g roup w as more l i ke l y de r i ved f rom p i pe ra l ean ances t o r s . A n ex t r emev i e w h a s b e e n t a k e n b y C u s s e t a n d C u s s e t ( 1 9 8 8 b ,c ) , w h o p r o p o s e d t h e P o d o s t e m o p s i d aas a c l a s s o f ang i osp e rms eq u i va l en t in r ank t o m ono co t y l edo ns and d i co t y l edons . Thus ,even a f t e r more t han a cen t u ry and a ha l f , t he r e l a t i onsh i ps o f r i ve r -w eeds r ema i nunreso l ved a t nea r l y eve ry h i ghe r l eve l o f c l a s s i f i ca t i on .

The phy l ogene t i c pos i t i on o f t he unusua l f ami l y H ydros t achyaceae i s a l so o f i n t e r e s l ,because t hese h i gh l y mod i f i ed p l an t s o f r i ve r r ap i d hab i t a t s have o f t en been a l l i ed w i t hP o d o s t e m a c e a e . T u l a s n e ( 1 8 4 9 ) , W e d d e l l ( 1 8 7 3 ) a n d B e n t h a m a n d H o o k e r ( 1 8 8 0 )r e c o g n i z e d th e H y d r o s t a c h y a c e a e as a s u b o r d i n a t e ta x o n o f P o d o s t e m a c e a e c o r r e s p o n d -i ng t o e i t he r subfami l i a l o r t r i ba l r ank . H u t ch i nson (1926) un i t ed t he f ami l i e s H ydro -s t achyaceae and Podos t emaceae i n t he o rde r Podos t ema l es . O t he r au t hor s ( e . g . Eng l e r .1930 : Takh t a j an , 1980 : C ronqu i s t , 1981 ; D ah l g ren , 19 89 )ha ve dou b t ed the i r r e l a ti on -


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8 D.H. Les et al. /Aquatic Botany 57 (1997) 5-27

o o 0 ^ _ o ~ = . ~ = ~ ~ .~ ~ o . = ~~ . ~ ~ - ~ ~ .~ ~ " ~

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D.H. Les et al ./Aq ua tw Botany 57 (1997) 5- 27 9

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10 D.H. Les et al./Aquatic Botany 57 (1997) 5-27sh ip , and hav e sugg es ted p lacem ent o f Hyd ros tachyac eae am ong va r ious fami l i e s in thesubc la ss A s te ridae , a re su l t cons i s t en t w i th a p re l imina ry ana lys i s o f r b c L d a t a ( H e m p e let al., 1995).

Molecu la r da ta have been app l i ed on ly spa r ing ly to the sys tema t ic s o f Po-dos temaceae . Seve ra l m ic romo lecu la r (phy tochem ica l ) s tud ie s have been conduc ted(e .g . , Burkhardt e t a l . , 1992; Romo Contreras e t a l . , 1993) , but these have essent ia l lybeen inconc lus ive fo r c l a r i fy ing the re la tionsh ip o f Pod os temaceae to o the r ang iospe rms ,inc lud ing Hydro s tachya ceae (Scog in , 1992). M acrom olecu la r (i .e . D NA ) d a ta a re v i r tu -a l ly non-ex i s ten t fo r Podos temaceae , bu t have y ie lded va luab le ins igh t s in to phy loge -net ic re la t ionships a t essent ia l ly a l l taxonomic levels in o ther angiosperms (Sol t is e t a l . ,1992) . The re a re now more than 2000 sequences ( rep resen t ing mos t ex tan t s eed p lan tfami l i e s ) fo r the ch lo rop la s t enco ded gen e r b c L i n G e n B a n k ( a D N A s e q u en c e d a ta b a s eacces s ib le v ia the In te rne0 . Indeed , r b c L da ta have been an impor tan t re source fo rinves t iga t ing high er level phy loge net ic re la t ionships in angiospe rm s (Chase e t a l. , 1993) .Comprehens ive s tud ie s o f the Sax i f ragaceae and Rosaceae (So l t i s e t a l . , 1993 ; Morganet a l. , 1994) as wel l as a sequence fro m Hy dro s tach yac eae (He m pel e t a l ., 1995) haveprov ided r b c L sequences fo r a tho rough sampl ing o f t axa deemed c r i t i ca l in theques t ion o f re la tionsh ips o f Podos tem aceae .

A s t u d y o f r b c L sequence da ta tha t inc ludes Podos temaceae p resen t s an inva luab leoppor tun i ty to inves t iga te and pe rhaps c la r i fy the re la t ionsh ips o f th i s b iza r re an -g iospe rm fam i ly . S imi la r s tud ie s have he lped to c la r i fy a va r ie ty o f sys tema t ic ques t ionsin s eve ra l aqua t i c p lan t g roups where reduc t ion and s t ruc tu ra l d ive rgence have compl i -ca ted effor ts to ascer ta in re la t ionships (e .g . Les e t a l . , 1991, 1993; Les and Haynes ,1995).In th i s s tudy , we repor t on a phy logene t i c ana lys i s o f r b c L sequences f rom e igh tspec ie s rep resen t ing e igh t d i f fe ren t gene ra o f Pod os temacea e s.l. These s equences haveb e e n a n a l y s e d a l o n g w i t h t h o s e f r o m m o s t m a j o r g r o u p s o f a n g i o s p e r m s w i t h a nemp has i s on t axa tha t have been sugges ted p rev ious ly a s pos sib le re la t ives o f f ive r -weeds .As fa r a s we a re aware , th i s s tudy i s the f i rs t u se o f molecu la r (DNA ) da ta , and the f ir s texp l i c i t u se o f c l ad i s t i c approaches to eva lua te the phy logene t i c pos i tion o f r ive r -weedsa m o n g a n g io s p e rm s .

2 . M e t h o d s

W e r e t r i e v e d f r o m G e n B a n k 8 2 r b c L sequences o f t axa f rom fami l i e s pu ta t ive lya l l i ed to the Podos temaceae in fo rmer t axonomic t rea tmen ts a s we l l a s ang iospe rm

F i g . 1 . C l a d o g r a m o f a n g i o s p e r m s a n d n o n - f l o w e r i n g s e e d p l a n t s c o n s t r u c t e d f r o m rbcL s e q u e n c e d a t ai n d i c a t e d a b a s a l p o s i t i o n o f t h e P o d o s t e m a c e a e a m o n g a n g i o s p e r m s . A l l b r a n c h l e n g t h s a r e p r o p o r t i o n a l .T opo logy shown i s one o f 160 equa l ly pa r s im onious t r e e s (3840 s t eps ) r e cove red i n t h i s ana lys i s . T he s t r i c tc o n s e n s u s t r e e d i f f e r e d b y o n l y m i n o r d e t a i l s ( s e e R e s u l t s ) . T h e p l a c e m e n t o f P o d o s t e m a c e a e i s p r o b a b l y aroo t ing a r t i f a c t c aused by l ong b ran ch a t t r a c ti on t o t he d ive rgen t non- f lo wer in g seed p l an t ou tg roups .H y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e a r e a d j a c e n t g r o u p s.


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D . H . L e s e t a l. / A q u a t i c B o t a n y 5 7 ( 1 9 9 7) 5 - 2 7 l 1

r C ~ d u s ~ C h a m a e c y p a d s

laG e u mo t e n t l l l aF r l ( 3 l r l l

- - H o I o d l s c u s- - S p i r a e a- - p r u n u s- - N e v i u s l aR h o d o t y p o s. C r a t a e g u s P h o U n la, $ o r b u s. K a g e n e c k la- N i i l ll a- - P d n s e p i a- E x o c h o r d a, S o r b a n a- A d e n o s to m a- - p u n m h l aR h a m n u s

C u c u r b i t a] , . m r - - - - E u o n y m u s' P l m a s s l aI ~ u P o l y g a l a~ P e l a r g o n i u mA s t i l b o i d e sD a r m e r aM u k d e m aH e u c h e r a

I I B ~ SaxifragaI ~ k a n a ~ i ~ a g aB o y k i m aS u l l i v a n t J aT e l e s o m x , OTHER

A N G I O S P E R M S~ D Hlppun$C a l l i t r l c h eI g l t a l l sJ I - - " 1 ~ A n t l r d l in u mU t r i c u l a r l a] r ~ - - - -" L " - ~ ' -- - - o l . . .S a n i c u l aJ l ~ o a b ~ n r ~ iucuba u c o m m l aC o m U ~ mC a m p t o t h e c a/ I I .r- HydrangeaP h i f a d e l p h u s5 c h i s m o c e r p u s

L o a s a P l s t l aI r - - I ~ A p o n o g e t o nA c o r u s~ Pel~mmla$11ututusAllarom

C h l o r a n t h u sC e r a t o p h y l l u mB r a s e n l aR a n u n c u l u sN e p e n t h e sR h e u mG u n n e r a D i a n t h u sK a l a n c h o e

C r a s s u l aI I - '1 ' M y d o p h y l lu m- - I ' H Yd~o~oasChYmSm ~ " H Y D R O S T A C H Y A C E A EManlth~mV a n r o y e n e l l aa . , ~ P O D O S T E M A C E A EO s e r y aM o u r e r aL F - ; E h . _I ~ G G n e tu mn e t u mI I W e l w i t s c h l aE p h e d r a[~ -- ~ E p h e d r a N O N - F L O W E R I N GS E E D PLANTS


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12 D.H. Les et al. /Aquatic Botany 57 (1997) 5-27f a m i l ie s f r o m a w i d e s a m p l in g o f m o n o c o t y l e d o n s a n d d i c o t y l e d o n s (T a b l e 1 ). W e a l sor e t r i eved 11 non- f l ow er i ng seed p l an t s equences t o i nc l ude a s ou t g roups . Trea t men t s o ft he Sax i f r agaceae and R osaceae (So l t i s e t a l . , 1993 ; Morgan e t a l . , 1994) w ere used t ogu i de t he i n i t i a l s e l ec t i on o f ' exempl a r s ' f r om t hese l a rge f ami l i e s , t o r ep resen t t hema j o r c l ades i den t i f i ed i n t hose s t ud i e s . I n add i t i on , w e sequenced rbcL fo r Apinagiayguazuensis C h o d a t e t V i s c h e r ( 1 3 4 8 n u c l e o ti d e s ), Cladopus austrosatsumensis ( K o i d z . )O h w i ( 1 3 4 9 n u c l e o t i d e s ) , Marathrum rubrum N o v e l o & P h i l b r i c k ( 1 3 5 4 n u c l e o t i d e s ) ,Mourera asperau (B ong . ) Tu l . ( 1169 nuc l eo t i des ) , Oserya coulteriana Tul . ( 1348nuc l eo t i des ) , Podostemum ceratophyllum Mi chx . ( 1348 nuc l eo t i des ) , Tristicha trifaria( B o r y e x W i l l d . ) S p r e n g e l ( 1 4 0 2 n u c l e o t i d e s ) , a n d Vanroyenella plumosa N o v e l o a n dP h i l b r i c k ( 1 3 4 8 n u c l e o t i d e s ) . T o i m p r o v e r e p r e s e n t a t i o n o f s o m e a n g i o s p e r m t a x a , w ea l so s equenced rbcL f r o m Aponogeton elongatus F . M u e l l . e x B e n t h . ( A p o n o g e -t onaceae ; 1183 nuc l eo t i des ) and Spirodela intermedia W . K o c h ( L e m n a c e a e ; 1 3 48n u c l e o ti d e s ). M e t h o d s f o r D N A i so l a ti o n , a m p l i f ic a t io n a n d s e q u e n c in g f o l l o w e d t h o s er epor t ed by L es e t a l. (1993) . A com pl e t e li s t o f t axa and acces s i on num ber s i s p rov i dedi n Tab l e 1 .

The f i na l da t a s e t r ep resen t ed 1403 nuc l eo t i des fo r t he 103 t axa compared . I ncom-p l e t e s ec t i ons o f s equences w ere coded a s mi s s i ng da t a . Phy l ogene t i c ana l ys i s o f t hes e q u e n c e d a t a w a s c o n d u c t e d u si n g P A U P v e r s io n 3 .1 .1 ( S w o f f o r d , 1 9 9 3 ) t o p e r f o r m a nu n w e i g h t e d m a x i m u m p a r s i m o n y a n a l y s i s . W e e m p l o y e d S I M P L E a d d i t i o n s e q u e n c e ,C O L L A P S E , M U L P A R S , S T E E P E S T D E S C E N T a nd T B R b r a n ch -s w a pp in g o p ti on s ina l l ana l yses . S t r i c t consensus w as used t o a s ses s r e su l t s w here mul t i p l e equa l l y mi n i ma ll eng t h t r ees w ere r ecove red . We se l ec t ed one r ep resen t a t i ve t r ee f rom each ana l ys i s t odep i c t t he r e l a t i ve b r anch l eng t hs o f t axa .F o u r a n a l y s e s w e r e c o n d u c t e d . T h e f ir s t an a l y si s d e s i g n a te d t w o c y c a d t a x a (Zamia,Cycas) as ou t g roups t o d i r ec t t he roo t i ng o f t he r ecove red t opo l ogy . B ecause t he r e su l t so f t h is an a l ys i s i nd i ca t ed po t en t i a l p rob l ems w i t h a t t r ac ti on o f l ong i ng ro up b r anch es byt he ou t g rou p ( s ee D i scus s i on) , w e a l so pe r fo rm ed a s e ri e s o f ana l yses t ha t exc l u ded t axaof p rogres s i ve l y d i s t an t r e l a t i onsh i p t o ang i ospe rms . The second ana l ys i s r e t a i ned t hes a m e a n g i o s p e r m s e q u e n c e s , b u t i n c l u d e d o n l y t h r e e s e q u e n c e s f r o m t h e d i v i s i o nG ne t ophy t a , w h i ch r e so l ved a s t he c l oses t s i s t e r g roup t o ang i ospe rms i n t he f i r s tana l ys is . Th e t h i rd ana l ys is exc l ud ed a ll non- f l ow er i ng p l an t s and used Ceratophyllumt o r oo t t he t r ee o f ang i ospe rm sequences a s sugges t ed by r e su l t s f r om a p r i o r ' g l oba l 'ana l ys i s o f s eed p l an t rbcL sequenc es (C hase e t a l. , 1993). Th e four t h ana l ysi s exc l ude dCeratophyllum, a ll m a g n o l i id , a n d a l l m o n o c o t y l e d o n a n g i o s p e r m s e q u e n c e s. T h er e s u l t i n g n e t w o r k w a s r o o t e d u s i n g Ranunculus, w h i c h f e l l a m o n g t h e b a s a l g r o u p o f' eud i co t ' t axa i n t he g l oba l rbcL analys i s (Chase e t a l . , 1993) .

I n t h e f o u r t h a n a l y s i s , r a n d o m t a x o n a d d i t i o n ( 3 5 r e p l i c a t e s ; M U L P A R S O N ;S T E E P E S T D E S C E N T O F F ) w a s u s e d t o s e a r ch f o r th e p r e s en c e o f m u l ti p le i s la n d s o f

Fig. 2. Histograms of branch lengths for fo ur analyses of rbcL sequence data. The branches leading to thePodostemaceae and Hydrostachyaceaeare extremely lon g and susceptible to attraction fro m other longbranches in all four analyses. The relative branch lengths of Podostemaceaeand Hydrostachyaceae h ifted asdistant outgroups we re excluded rom the analyses (analysis sequence 1-4).


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D.H. Les et al. /Aquatw Botany 57 (1997) 5-27 13

15 ,

> 1 0 ,o",=

0 7

A n a l y s is # 1

. . J , I , , J hPODOSTEMACHEAy~ROSTACHYACEAEv vI l l l l i l i ~ l i ~ l l ~ J I , I I h l , , , , ,15 ass igned branch leng ths 7"3

] J A n a l y s i s # 2

O O a ss ig n e d b r a n ch l e n g th s 7 3

i s ] A n a ly s is # 3l , l l k l , , HYDROST ACHYACEAEJ= 5 YI I I I P O D O S T E M A C E A E

0 0 ass igned branch leng ths 73

1 5 ] h A n a l y s i s # 4| ~ h , , , I . .o ~ o s , . 0 . . . c ~ , ~I I l l l l l l i l I l l I v P O D O S T E M A C E A E

0 ass igned branch leng ths 71


10/23

14 D .H . Le s e t al . / A q u a t i c B o t a n y 5 7 (1 9 97 ) 5 - 2 7

minim a l l eng th t rees (Madd ison , 1991). W e co mp uted boo t s trap suppor t (Fe ls ens te in ,1985) fo r the ind ica ted m ono phy ly o f c lades us ing 200 rep l i cate s (SIM PL E add i t ions e q u e n c e ; M U L P A R S O F F ; S T E E P E S T D E S C E N T O F F ) . A n u n c o r r e c t e d e s t i m a t e o fsequence d ive rgence among Podos temaceae spec ie s was ob ta ined by compar ing a 1348nuc leo t ide reg ion su rv eyed fo r a l l spec ie s (excep t Mourera where the resul ts a re basedon 1169 s ites ). Th e dis t r ibut ion of branc h lengths w as exa m ined for a representa t ive t reein each ana lys i s to p rov ide an ove rv iew of re la t ive b ranch l eng ths fo r Podos temaceaeand o the r g roups .

3 . Re s u l t s

From our ana lys i s o f the comple te da ta s e t us ing cycad s equences fo r ou tg rouproot ing, w e recov ered a to ta l of 160 t rees (3840 s teps ; con s is tency index, C I = 0 .28,0 .25 exclu ding u ninf orm at ive charac ters ; re tent ion index, R I = 0 .62; Fig . 1). Thistopo logy showed the Podos temaceae a s basa l to o the r ang iospe rms , bu t re so lved theingroup topo logy in a h igh ly i r regu la r fa sh ion . Hyd ros tachy aceae were the c loses t g roupto Podos temace ae , fo l low ed by H a lo ragaceae , Cras su laceae and Gu nneraceae whichwere basa l to o the r ang iospe rms . Succes s ive ly , the Sax i f ragaceae and Rosaceae ap -pea red nex t wi th mem bers o f the Corna le s , subcla s s As te r idae , subc la s s Caryoph y l l idae ,subc la ss Mag no l i idae and m ono co ty led ons de r ived u l t ima te ly (F ig . 1 ). The topo logydep ic ted in th i s ana lys i s , where monoco ty ledons and magno l i id d ico ty ledons wereamong the g roups tha t appea red to be mos t h igh ly de r ived , re sembled an inve r tedve rs ion o f re la t ionsh ips tha t a re gene ra l ly a s sumed fo r ang iospe rms (e .g . Cronqu is t ,1981), o r tha t have been ob ta ined in s im i la r ana lyses exc lud ing Podos tem aceae t axa (e .g .Chase et al . , 1993).

The b ranch l ead ing to the Po dos tem aceae (40 s t eps ) was the 16 th longes t o f the 203branches re so lved in the c ladogram; the b ranch to H ydros tach yaceae (50 s t eps ) wasten th longes t (F ig . 2 ) . In pe rspec tive , the b ranch s epa ra ting mo noco ty ledon s f romdico ty ledons was on ly 21 s t eps , tha t s epa ra t ing the ingroup (ang iospe rms ) f rom theou tg roup (non - f lower ing p lan ts ) was 53 s teps , and tha t s epa ra t ing Tristicha f r o m o t h erPodo s temaceae was 43 s teps . The s t r ic t consensus t ree was v i r tua l ly iden t i ca l to the onepresen ted , and d i f fe red e s sen t i a l ly by the co l l apse o f some nodes wi th in the Sax i f ra -gaceae ( Astilboides, Bergenia, Darm era, M ukdenia) a n d P o d o s t e m a c e a e ( Apinagia,Marathrum, Vanroyenella) .

F i g . 3 . C l a d o g r a m c o n s t r u c t e d f ro m r b c L d a t a f o r v a r i o u s g r o u p s o f a n g i o s p e r m s u s i n g t h r e e o u t g r o u p g e n e r af r o m t h e d i v i s io n G n e t o p h y t a t o r o o t t h e n e t w o r k . A l l b r a n c h l e n g t h s a r e p ro p o r t io n a l . T h e t o p o l o g y s h o w n i so n e o f 8 0 e q u a l l y p a r s i m o n i o u s t r e e s ( 3 4 5 8 s t e p s ) r e c o v e r e d i n t h i s a n a l y s is . A g a i n , t h e s t ri c t c o n s e n s u s t re ed i f f e r e d b y o n l y m i n o r d e t a i l s ( s e e R e s u l t s ) . T h e p o s i t i o n o f P o d o s t e r n a c e a e r e m a i n e d b a s a l t o o t h e ra n g i o s p e r m s i n t h i s a n a l y s i s , b u t t h e t o p o l o g y o f o t h e r a n g i o s p e r m s a l s o r e m a i n e d ' i n v e r t e d ' r e l a t i v e t or e l a t i o n s h i p s t y p i c a l l y p e r c e i v e d b y c o n t e m p o r a r y t a x o n o m i s t s . P h y l o g e n e t i c a l l y , t h e H y d r o s t a c h y a c e a e ,H a l o r a g a c e a e ( M y r i o p h y l l u m ) a n d C r a s s u l a c e a e w e r e t h e c l o s e s t a n g i o s p e r m f a m i l i e s to t h e P o d o s t e m a c e a e i nt h i s ana l ys i s .


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D . H . L e s e t a l. / A q u a t i c B o t a n y 5 7 ( 1 9 9 7) 5 - 2 7 1 5

;eum' R O S A C E A E

. - - - M I S C . E U D I C O T S

~ . G A C E A E

~ ! r r h H ippurls '~1al l i tr iche /gitalls /mum iculada /. . . . J - M I S C . A S T E R I D S/desla /

JI I . J ~ A l a n g i u m II I r - [J - '~ c a m p ~-eca INylmaI H I " C O R N A L E SI I I ~ Phl ladelphus II ~ SchlsmocarpusI ~ Loasa -- -J~ ~ ~ ~ I ~ ~ T ' 3 M O N O C O T Y L E D O N S_J ' Acorns, ~ - ~ 4 ~ S . . . . P eperm la m 1

I ] ' AsarumL I ~ M agn ia ~ " M A G N O L I I D S-- .J I ~ Chloranthu$ /I I . ~ C e r a t o p h y l l u m /. - I- 1eum C' , spiRhcelUm~ A R Y O P H Y L L I D Si DmnthusGunnera. ~ ~ M y K a la nc hesedum I - C R A S S U L A C E A ECrassulariophyllum,y d ro .~ .c ,y . m H Y D R O S T A C H Y A C E A EI j - Marathrum~ LVanroyenel la |,~LApineg a |PodoetemonV ' t ~ _ ~ c l a a o p . , I " P O D O S T E M A C E A Eu o ser ya /.~-..--Meurera /TrlstJcha Gnetum ~ , .I I W e lw it sc h la G N E T O P H Y T AEphedra


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16 D .H . Le s e t a l. / A q u a t i c B o t a n y 5 7 ( 19 9 7) 5 - 2 7

Lim i t ing the ou tg roup to th ree Gn e tophy te s equences ( s econd an a lys i s) re su l t ed in 80trees (3458 s teps ; CI --- 0 .30, 0 .26 exclu ding u ninfo rm at ive charac ters ; RI = 0 .60). Thisana lys i s (F ig . 3 ) re ta ined the pos i t ion o f Pod os temacea e a s basa l to o the r ang iospe rms ,aga in wi th H ydros tachy aceae , Ha lo raga ceae and C ras su laceae a s the succes s ive ly c loses tang iospe rm fami l i e s (F ig . 3 ) . De ta i l s o f th i s topo logy d i f fe red l i t t l e f rom those o f thef i rs t analys is (Fig . 1) . The branch leading to Podos temaceae (32 s teps) was the 23rdlonges t amo ng the 188 b ranches in th i s ana lys is (F ig . 2 ) ; the rem a inde r o f the ingroupwas suppor ted by a b ranch o f 38 s t eps . O the r b ranch l eng ths fo r re la t ive compar i sonwere : mo noco ty ledon s (21 s teps ), ou tg roup (90 s teps ), H ydros tach yaceae (50 s t eps ) andTristicha/other Podo s temaceae (42 s teps ). The s t ri c t consensus t ree d i f fe red on ly by theco l l apse o f some nodes in the Rosaceae (Prinsepia/Exochorda, Neviusia/Rhodotypos),Sax i f ragaceae (Asti lbo ide s, Be rg e n ia , D arm e ra , M uk de n ia ) a n d P o d o s t e m a c e a e( Apinag ia, M arathrurn, Vanroyenella).

The th i rd ana lys i s , wh ich exc lude d a l l non- f low er ing p lan t s equences and u sedCeratophyllum to roo t the rema in ing s equences , gene ra ted 8300 t rees be fo re reach ingthe t ree buf fe r ove r f low im posed by co mp ute r mem ory l imi ta tions . Thus , th i s s ea rch wasposs ib ly ine f fec t ive a t e s t ima t ing the m in ima l l eng th topo logy . The t rees recove red were3148 s teps in length (C I = 0 .31, 0 .26 exclu ding unin form at ive charac ters ; RI = 0.61) .S t r i c t consensus o f these t rees (no t shown) p roduced a topo logy compa t ib le wi th theresu l ts o f s imi la r ana lyses (e .g . C hase e t a l . , 1993), where m onoc o ty ledon s andm agno l i id d ico ty ledon s occured in a pos i t ion basa l to a ' eud ico t ' c l ade . The po s i t ion o fPodos temaceae was poor ly re so lved , bu t nes ted wi th in a c lade tha t inc luded va r iousmembers o f subc la s ses As te r idae and Caryophy l l idae . A l though fa r l e s s re so lved thanthe exam ple p resen ted (F ig . 4 ) , the s t r ic t consensus t ree a l so dep ic ted the Hy dros tachy-aceae a s the s i s t e r g roup to Podos tem aceae . Ranuncu laceae and Gunneraceae w ere basa lto the ' eud ico t ' c l ade tha t con ta ined the members o f Podos temaceae .

The b ranch suppor t ing the Podos temaceae (63 s t eps ) rema ined re la t ive ly long ( f i f thlonges t o f the 179 b ranches in th i s ana lys i s ; F ig . 2 ) and ind ica ted the cons ide rab led ive rgence o f th i s fam i ly f rom the o the r ang iospe rms wh ose s equences were inc luded inthe analys is . Branch lengths for re la t ive comparison were : outgroup (65 s teps) , Tris-t icha/o ther Podo s temaceae (43 s t eps) , mon oco ty led ons (22 s t eps ) and ' eud ico t s ' (17s teps ). The b ranch suppor t ing the Hyd ros ta chy ace ae /P od os tem ace ae c lade was 43 s t eps.

The four th ana lys i s ( re s t r i c t ed to ' eud ico t ' s equences and roo ted by Ranunculus)p r o d u c e d 1 6 0 t r e e s ( 2 5 3 5 s t e p s ) w h i c h s h o w e d t h e H y d r o s t a c h y a c e a e / P o d o s t e m a c e a eas a s i s te r c l ade to the subc la s s Caryo phy l l idae ( re su lt s no t shown) . H owev er , 35random taxon add i t ions recove red an i s l and o f shor te r t r ees (2534 s t eps ) tha t d i f fe redcons ide rab ly in topo logy . Here , too , the ana lys i s was im pa i red by a t ree -buf fe r ove r f low.

F i g . 4 . C l a d o g r a m c o n s t r u c te d f r o m r b c L s e q u e n c e s o f v a r i o u s a n g i o s p e r m s u s i n g CeratophyUum t o roo t t hene twork . Al l b ranch l eng ths a re p ropor t i ona l . T h i s t opo logy i s one o f 8 ,300 equa l ly pa r s im onious t r e e s (3 ,148s t eps ) r e cove red . T he s t r i c t consensus t r e e was s im i l a r ove ra l l t o t he r e su l t shown, bu t con t a ined a g rea t e rp r o p o r t i o n o f u n r e s o l v e d n o d e s . H e r e , P o d o s t e m a c e a e f e l l w i t h i n a c l a d e c o m p r i s i n g H y d r o s t a c h y a c e a e a n dfam i l i e s o f t he o rde r Corna l e s , a l t hough th i s r e su l t m ay rep re sen t a subop t im a l so lu t i on ( see Di scuss ion) . Othe ra n g i o s p e r m g r o u p s o c c u p i e d p o s i t i o n s t h a t a r e r e a s o n a b l y c o m p a t i b l e w i t h c o n t e m p o r a r y p h y l o g e n e t i ch y p o t h e s e s .


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D .H . Le s e t a l . / A q u a t i c B o t a n y 5 7 ( 19 9 7) 5 - 2 7 1 7

nGe u m. _J ~ - - - - - Ru b u sI [ ~ r - - - P o t en t ll la~ F ra g a n ar l L - - " - " - - - F llip e n du la~ HoIod iscus~--~~ Sp i raea[ ~ Prunus~ Pr insep laI ~ E xo ch o rd a " R O S A C E A ENeviusiar ] - I ~ - - R h o d o ty p o sI I _ j - Cra t a e g u sII J I' - PhotJniaII ,---IL-- SorbusI ' - " 1 1 ~ L K a g e n e ck i a1 I l l b - s o r b a ~ aI I "1 L -Adano~ toma

I L ~ P u r s h i aRh a m n u s E u o n ym u sI I , ~ 1 ' P am a ss la I - M I S C . E U D I C O T SI r - - i I F o l i a t e /I I I ~ Nothofagus |I ~ B e g o n i a /I ~ Cucu rb i ta /I . P e la rg on lu m~ _ . T e y k i ni aSul l lvant ia |l e so m x

I '~ _ j - - - -H e u c h e r a II ~ " T~aeaxl~aga I" SAXIFRAGACEAEI 1 - ; . . . . S " r a " Ir - - ' l P B e m e nia /I I ~ Mukd en la /. .J I - - Ast l l bo ldes K a l a n ch o e~ S ed u m 7 = C R A S S U L A C E A EI I / Crassu la ~1 1I Myr iophyl l um (HALORAG ACEAE)

nmyene l la |o d o s t e m o m |n ag la C la dp us ! , P O D O S T E M A C E A E~ / a /

- jH yd ro 6ta ch ys - - H Y D R O S T A C H Y A C E A EC o m u sI- ~ A iang lum~ Ca m p t o t h e ca_ 1 ~ , , C O R N A L E S~ Hyd ra n g e aL . [ 1 - ~ P h l l a d e l p h u sS ch l sm o caL o a sa

~ Solanum M ISC . EU DIC O TSP~lcuba- - . [ " ~ G a r r ya E u c o m r nl af- Bam~l~laNepenthes ~SpinRa e u C A R Y O P H Y L L I D SD l a n t h u s . , , IGu n n e rs (GUNNE RA CE A E )Ra n u n cu l u s (RA NUNCUL A CE A E ), Peperorn ia (P IPERACEAE)I S a u m ru a (S A URURA CE A E ) I -A s a ru m (A R IS T O LO C H IA C EA E ) j M A G N O L I I D SBrasor l l e (NYMPHAEACEAE)P I s U a " 1i I Splrod ela L

A po no go to n j M O N O C O T Y L E D O N SA co ru sM a g n on s ( M AG N O L IA C E AE ) ~ M A G N O L I I D SCh l o ra n t h u s (CHL ORA NTHA CE A E )Ce ra t o p h y lt u m (CE RA TOP HY L L A CE A E )


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18 D.H. Les et al. /Aquatic Botany 57 (1997) 5-27

T h e t o p o l o g y o f t h e 6 4 0 t r e e s r e c o v e r e d i n t h i s i s l a n d ( 2 5 3 4 s t e p s ; C I = 0 . 3 5 , 0 . 2 9e x c l u d i n g u n i n f o r m a t i v e c h a ra c te r s; R I = 0 . 6 4 ) j o i n e d H y d r o s t a c h y a c e a e a n d P o -d o s t e m a c e a e a s s i st e r ta x a a n d p l a c e d t h e t w o f a m i li e s i n a c l a d e w i t h C r a s s u la c e a e a n dH a l o r a g a c e a e . T h e s e f a m i l ie s n e s t e d i n a c l a d e t h a t c o m p r i s e d S a x i f r a g a c e a e , R o s a c e a e ,

24 %

15%


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D.H. Les et al./Aquatic Botany 57 (1997) 5-27 19a n d a v a r i e t y o f e u d i c o t f a m i l i e s ( F i g . 5 ). T h e s t r i c t c o n s e n s u s s h o w e d m u c h t h e s a m et o p o l o g y w i t h l e s s r e s o l u t i o n a s n o t e d f o r c e r t ai n m e m b e r s o f P o d o s t e m a c e a e , R o s a c e a ea n d S a x i f r a g a c e a e i n p r e v i o u s a n a l y s e s ; r e s o lu t i o n o f s e v e r a l o t h e r c l a d e s w a s s l ig h t l yr e d u c e d i n th e c o n s e n s u s t r e e, b u t t h e m a j o r p o r t i o n s o f t he t o p o l o g y d i d n o t d i f f e rf u n d a m e n t a l l y f r o m t h a t p r e s e n t e d i n F i g . 5 .

T h e P o d o s t e m a c e a e b r a n c h ( 5 3 s t e p s ) w a s t h e s ix t h lo n g e s t o f t h e 1 65 b r a n c h e s ( F i g .2 ) ; Tristicha w a s s e p a r a t e d fr o m o t h e r P o d o s t e m a c e a e b y a b r a n c h l e n g t h o f 4 3 s t e p s.T h e H y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e w e r e j o i n e d b y a b r a n c h o f 3 4 s te p s . T h eb r a n c h s u p p o r t i n g a c l a d e w it h H y d r o s t a c h y a c e a e , P o d o s t e m a c e a e , H a l o r a g a c e a e a n dC r a s s u l a c e a e w a s 2 0 s t e p s . B o o t s t r a p s u p p o r t f o r s e v e r a l d e e p i n t e r n a l n o d e s w a s l o w( < 5 - 9 % ) , i n c lu d i n g th e a s s o c i a t io n o f P o d o s t e m a c e a e a n d H y d r o s t a c h y a c e a e w i thC r a s s u l ac e a e a n d H a l o r a g a c e a e ( 8 % ) . S u p p o r t fo r th e m o n o p h y l y o f P o d o s t e m a c e a e w a sh i g h ( 1 0 0 % ) , b u t s u p p o r t fo r an a s s o c i a t io n w it h H y d r o s t a c h y a c e a e w a s m u c h l o w e r( 3 4 % ) . M o n o p h y l y o f t h e C r a s s u l a c e a e , R o s a c e a e , a n d S a x i f r a g a c e a e ( e x c l u d i n g Par-nassia) w a s a l s o s t r o n g l y s u p p o r t e d , w i t h b o o t s t r a p v a l u e s o f 9 6 , 9 0 a n d 1 00 e~r e s p e c t i v e l y ( F i g . 5 ) .

T h e rbcL s e q u e n c e o f Tristicha w a s 6 . 8 - 8 . 6 % d i v e rg e n t f r o m s e q u e n c es o f o th e rP o d o s t e m a c e a e g e n e r a ( T a b l e 2 ) . T h e rbcL v a r i a t i o n a m o n g t he r e m a i n i n g f i v e g e n e r ao f P o d o s t e m a c e a e s u r v e y e d ra n g e d f r o m 0 .7 t o 3 . 2 % , w i t h t h e h i g h e s t le v e l o b s e r v e db e t w e e n t h e s e q u e n c e s o f Cladopus a n d Mourera ( T a b l e 2 ).

4 . D i scuss ion

T h e rbcL d a t a i n d ic a t e a s u b st a n ti a l d e g r e e o f s e q u e n c e d i v e r g e n c e b e t w e e n m e m -b e r s o f t h e P o d o s t e m a c e a e t h a t w e e x a m i n e d . S e q u e n c e d i v e r g e n c e b e t w e e n Tristichaa n d o t h e r P o d o s t e m a c e a e e x c e e d e d t w i c e th e l ev e l o f d i v e r g e n c e o b s e r v e d a m o n g t hef iv e o t h e r g e n e r a o f P o d o s t e m a c e a e s u r v e y e d f r o m d i f f e re n t c o n ti n e n t s ( T a b l e 2 ).A l t h o u g h n o t d e f i n i t i v e , t h i s r e s u l t le n d s s u p p o r t t o t a x o n o m i c t r e a t m e n t s t h a t h a v em a i n t a i n e d T r i st i c h a c e a e a n d P o d o s t e m a c e a e a s s e p a r a t e f a m i l i e s. Tristicha w a s b a s a l t oo t h e r g e n e r a o f P o d o s t e m a c e a e i n al l a n a l y s e s , an d t h e m o n o p h y l y o f Tristicha a n do t h e r P o d o s t e m a c e a e w a s s u p p o r t e d b y 1 0 0% b o o t s t r a p v a l u e s.

I n a l l a n a l y s e s , a h i g h d e g r e e o f s e q u e n c e d i v e r g e n c e w a s e v i d e n t b e t w e e n b o t h t h e

Fig. 5. A cladogram of 'eud icot' taxa constructed from rbcL sequences using Ranunculusto root the netw ork.A ll branch lengths are proportional. The topology show n is one o f 640 equa lly parsimonious trees recovered(2534 steps) using 35 random input additions of taxa. The strict consensus tree was not as well resolved, butdepicted the m ajor features of the topo logy illustrated by this example. The level of internal support (bootstrapvalue) is indicated at each node (a few nodes lack values because of space limitations). The Podostemaceaereside in a clade along with the Hyd rostachyaceae as its im med iate sister gro up . The C rassulaceae andHaloragaceae occur as an adjacent clade. Although this clade lacked strong internal support (8% bootsrapvalue), the association of these families coincides with the opinions of several contemporary taxonomists.Other renditions of relationships are similar to tho se resolved in global analyses of rbcL data and are fairlyrepresentattve of traditional taxonomic perspectives. The Podostemaceae were strongly supported as amo nophyletic gr ou p in this ana lysis. Internal support for the assoc iation of H ydrostachyaceae and P o-dostemaceae (34% bootsrap value) was markedly reduced.


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20 D . H . L e s e t a l. / A q u a t i c B o t a n y 5 7 ( 1 9 97 ) 5 - 2 7

.r "0

o

0

o~

~'=~

I 0

0

I r . - 0

0 0

~.o. .

I0 0 0c S c S c S d d

d d ~ o d d

d


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D.H. Les et al. / Aquatic Botany 57 (1997) 5- 27 21

P o d o s t e m a c e a e a n d H y d r o s t a c h y a c e a e a n d o t h e r a n g i o sp e r m s . T h e b r a n c h s e p a r a ti n g t h eP o d o s t e m a c e a e c la d e f r o m o t h e r a n g i o s p e r m t a x a ( F ig s . 1 a n d 3 - 5 ) r a n g e d f r o m 3 2 - 6 3s t eps i n l eng t h and a l w ays occur r ed i n t he t op 12% of r epor t ed b r anch l eng t hs . I n a l lana l yses (F i gs . 1 and 3 -5 ) , t he b r anch l ead i ng t o t he Podos t emaceae w as subs t an t i a l l yl o n g e r t h a n t h a t w h i c h s e p a r at e d m o n o c o t y l e d o n s a n d d i c o t y l e d o n s . T h i s h i g h l e v e l o fs e q u e n c e d i v e r g e n c e i s c o m p a t i b l e w i t h t a x o n o m i c o p i n i o n s t h a t h a v e s u g g e s t e d t h esegrega t i on o f Po dos t em acea e a s an i so l a t ed o rde r , Podos t em a l es (e . g . D ah l g ren , 1980 ;Takh t a j an , 1980) . The b r anch l ead i ng t o H yd ros t ac hya ceae (50 s t eps ) w as on l y th r ees t eps l onge r t han t he ou t g roup b r anch i n t he ana l ys i s t ha t i nc l uded a l l t axa .

A na l ys i s o f rbcL da t a i nd i ca t e s t ha t t he ex t r eme morpho l og i ca l d i ve rgence o f t heP o d o s t e m a c e a e a n d H y d r o s t a c h y a c e a e c o r r e la t e s w it h a c o m p a r a b l e l e v e l o f d iv e r g e n c ea t the D N A sequen ce l eve l . Th i s t ype o f evo l u t i ona ry h i s to ry ( i. e . h i gh l y d i ve rgen tcha rac t e r s ) i s a s p rob l ema t i c f o r pa r s i mony ana l yses o f mol ecu l a r da t a a s i t i s w i t hmo rpho l og i ca l da t a. Th e d i ve rgenc e o f bo t h t ypes o f cha rac t e r s ma kes i t d i f fi cu l t t o tr acet he r e l a t i onsh i p o f t hese g roups w i t h any h i gh degree o f ce r t a i n t y , due t o a p rob l emknow n as l ong b r anch ' a t t r ac t i on ' (Fe l sens t e i n , 1978 ; H endy and Penny , 1989 ; O l ms t eadand Pa l mer , 1994) . S i mpl y s t a t ed , l ong b r anches c r ea t ed by cha rac t e r s t a t e s (mol ecu l a ro r m o r p h o l o g i c a l ) o f e x t r e m e l y d i v e r g e n t t a x a m a y c o n v e r g e t o a n i n c o r r e c t t r e et opo l ogy a s t he t r ue phy l ogene t i c s i gna l i n t he da t a becomes p rogres s i ve l y d i l u t ed .B e c a u s e t h e b ra n c h b e t w e e n t h e ' i n g r o u p ' a n d ' o u t g r o u p ' is g e n e r a ll y l o n g , o t h e r l o n gbranches w i t h i n t he i ng roup a r e suscep t i b l e t o r oo t ing e r ro r s , i. e. t hey ma y m ov e t he roo to f t he i ng roup by a t t r ac t i on o f l ong b r anches i n t he i ng roup and ou t g roup . I n p r ac t i ce , i th a s p r o v e n e x t r e m e l y d i f f i c u l t t o d e c i d e i n s u c h c a s e s w h e t h e r t h e p l a c e m e n t o f ad i ve rgen t t axon i s co r r ec t o r e r roneous .A dd i ng add i t i ona l t axa t ha t po t en t i a l l y cou l d b r eak up t he l ong b r anch i s oneso l u t i on , bu t no o t he r ex t an t f ami l i e s w i t h a po t en t i a l l y c l ose r e l a t i onsh i p t o e i t he rH y d r o s t a c h y a c e a e o r P o d o s t e m a c e a e h a v e b e e n i d e n ti fi e d .

The Ceratophyllum sequen ce i s a l so qu i t e d i ve rg en t and i s a l so suspec t a s a pos s i b lel ong b r anch (Q i u e t a l . , 1993) . H ow ever , t he a s soc i a t i on o f t axa us i ng t he Ceratophyl-lure roo t i ng i n ou r s econd ana l ys i s w as s i mi l a r t o t ha t o f t he l a rge r ana l ys i s o fa n g i o s p e r m rbcL sequences by Chase e t a l . (1993) . I t i s in teres t ing tha t the longb r,'m c he s o f P o d o s t e m a c e a e a n d H y d r o s t a c h y a c e a e d i d n o t c o n v e r g e w i t h Ceratophyllumi n any o f t he ana l yses .

We be l i eve t ha t ou r i n i t i a l ana l yses o f Podos t emaceae i l l u s t r a t ed p r ec i se l y t hep h e n o m e n o n o f l o n g b r a n c h a t t r a c t i o n w h e n n o n - f l o w e r i n g p l a n t s e q u e n c e s w e r e u s e df o r o u t g r o u p r o o t in g . T h o s e a n a l y s e s y i e l d e d a t o p o l o g y d e p i c t in g P o d o s t e m a c e a e a s abasa l angiosperm l ineage (Figs . 1 and 3) . Super f ic ia l ly , th i s resul t i s not ent i re lyi mpl aus i b l e , g i ven t he r ecen t sugges t i on t o r ecogn i ze t he Podos t emops i da a s a d i s t i nc tang i ospe rm c l a s s (C usse t and C usse t , 1988b , c ) . H ow ever , i n add i t i on t o ex t ens i vemo rpho l og i ca l s t ud i e s by J~ iger -Z ii m (1995 ) w h i ch d o no t supp or t t h is i n t e rp r e t a ti on , w ea re a l so r e l uc t an t t o accep t t he basa l pos i t i on o f Podos t emaceae i n t he l i gh t o f r e su l t sf r o m s u b s e q u e n t a n a l ys e s . M a n i p u l a ti o n s o f o u r d a t a s e t s h o w e d t h a t e x c l u s io n o f e v e n as i n g le o u t g r o u p s e q u e n c e ( e .g . f r o m G n e t o p h y t a ) i n o n e c a s e m o v e d t h e p o s i t io n o f th ePod os t em acea e c l ade t o r e so l ve a s a s i s te r g roup t o the G ne t o phy t a ( r e su l ts no t show n) .Th i s i s no t pa r t i cu l a r l y su rp r i s i ng (nor accep t ab l e t axonomi ca l l y ) , g i ven t he ex t r eme l y


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22 D.H. Les et al./Aquatic Botany 57 (1997) 5-27long b ranches tha t s epa ra te t axa in the Gne top hy ta , the c lade tha t re so lves a s the c loses tex tan t s i s te r g roup to ang iospe rms . Gnetum and Welwitschia were s epa ra ted by a b ranchof 65 s teps ; the c lade of Ephedra spec ie s by a b ranch o f 50 s t eps , and the Gnetum an dWelwitschia clade by a branch o f 35 s teps in the f i rs t analys is (Fig . 1). This aggreg at ionof long b ranches w ould be a l ike ly p lace fo r o the r long b ranches (e .g . Podo s temaceae ,H y d r o s t a c h y a c e a e ) to c o n v e r g e.

The l ab i l e phy logene t i c pos i t ion o f Podos temaceae was whol ly ev iden t in re su l t sf rom the ana lyses desc r ibed above . W i th d ive rgen t non- f lower ing p lan t s equencesinc luded in the ana lys i s , Podo s temaceae re so lved a s a basa l ang iospe rm c lade ; however ,in te r re la t ionsh ips wi th in the ang iospe rm ingroup were re so lved in a h igh ly i r regu la rfa sh ion tha t conf l i c t ed wi th mos t t axonomic op in ions . L imi t ing the ou tg roup to th reeGne tophy te s equences re ta ined the Podos temaceae in a pos i t ion basa l to o the r an -g iospe rms , bu t a l so re ta ined a topo logy o f ing roup re la t ionsh ips tha t was ' i nve r ted 'phy log ene t i ca l ly . Group s tha t have b een h ypo thes ized to rep resen t p r imi t ive t axa incoun t le s s sys tema t ic s tud ie s appea red in d e r ived pos i tions and v ice ve rsa . Th i s ou tcom eind ica ted tha t the long b ranches o f the non- f lower ing p lan t s equences re su l t ed in amisp laced roo t o f ang iospe rm sequences a t the long b ranches de l imi t ing the Hydro-s tachyaceae and Podos temaceae ra the r than in the v ic in i ty o f magno l i id t axa where theang iospe rm ro o t i s gene ra l ly a s sumed . N ever the le s s , the Cras su laceae and H a lo ragaceaereso lved a s the c loses t g roups to Podo s temaceae and Hy dros tachy aceae in these inve r tedtopologies (Figs . 1 and 3) .

L imi t ing the ana lys i s exc lus ive ly to ang iospe rms re su l t ed in the nes t ing o f Hydro-s tachyaceae and P odos tem aceae wi th in an a l l i ance o f a s te rid and ca ryop hy l l id g roups(e .g . Cornales , As terace ae , Cal l i t r ichaceae) , a pos i t ion tha t was fa i r ly rem ote f romHaloragaceae and Cras su laceae . We be l i eve tha t th i s re su l t may re f l ec t ou r fa i lu re tosearch for o ther , poss ibly shorter , i s lands of t rees during th is analys is . A s imilara s soc ia t ion o f t axa was recove red in the in i t i a l heur i s t i c s ea rch o f ana lys i s 4 , bu t asea rch fo r shor te r t rees d id recov e r a top o logy on e s t ep shor ter wh ich re -e s tab l i shed thea s s o ci a ti o n o f H y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e w i t h t h e H a l o ra g a c e a e a n d C r a ss u -laceae (F ig . 5 ) . These obse rva t ions fu r the r ind ica te the ex t reme ly l ab i l e pos i t ion o fH y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e i n rbcL ana lyses . T rees tha t d i f fe red by on ly as ing le s t ep in l eng th p laced these fami l i e s among as soc ia t ions o f ang iospe rms tha trep resen t ex t reme ly d i f fe ren t phy logene t i c a f f i l i a t ions ( i . e . subc la s s Ros idae ve rsusCaryophy l l idae ) .Ye t , even when we re s t r i c t ed the ana lys i s to ' eud ico t ' f ami l i e s (which re so lved ac lade cons i s t ing o f the H a lo ragaceae , Cras su laceae , Hyd ros tachyac eae and Po-dos temaceae ) , low boo t s t rap va lues rende red the mo nop hy ly o f th is c l ade deba tab le .The re w as on ly 8% boo ts t rap suppor t fo r th i s a s soc ia t ion (which few sys tema t i s ts wou ldrega rd a s compe l l ing) , and on ly 34% boo ts t rap suppor t fo r the a s soc ia t ion o f Hydro-s tachyaceae and Podos temaceae . We emphas ize tha t Hempe l e t a l . (1995) ob ta ined as imi la r l eve l o f boo t s t rap suppor t (30-34%) fo r the a s soc ia t ion o f Hydrostachys andDecumaria (Hydrangeaceae ) in ana lyses tha t d id no t inc lude members o f Po-d o s t em a c e a e . A l t h o u g h w e v i e w t h e r e la t io n s h ip o f H y d r o s t a c h y a c e a e a n d H y -drangeaceae a s h igh ly improbab le , we canno t p rov ide much be t t e r suppor t fo r thea s s o ci a ti o n o f H y d r o s t a c h y a c e a e a n d P o d o s t e m a c e ae .


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D.H. Les et al./Aquatic Botany 57 (1997) 5-27 23

In any even t , i t i s ev i den t t ha t r emovi ng t he l ong ou t g roup b r anches r e su l t ed i n t hep l a c e m e n t o f H y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e a s a s i s te r g r o u p t o th e C r a s s u l a ce a ea n d o t h e r r o s e a c o u s f a m i l i e s . W i t h o u t o t h e r p h y l o g e n e t i c a n a l y s e s o f t h e s e g r o u p s t os e r v e a s te s ta b l e h y p o t h e s e s , w e c a n o n l y r e s ta t e th e o p i n i o n s o f m a n y t a x o n o m i s t s w h oh a v e s u g g e s t ed t h e p l a c e m e n t o f t h e g r o u p s o m e w h e r e i n t h e v i c i n i ty o f t h e R o s a le s .

M o s t o p i n i o n s h a v e f a v o r e d a n a l l i an c e o f th e P o d o s t e m a c e a e t o e i t h e r S a x if r a g a c e a eor C ras su l aceae (C ronqu i s t , 1981), I n t h is r e spec t , ou r r e su l ts suppor t ea r l i e r hypo t heses .H o w e v e r , t h e l o w l e v e l o f b o o t s t r a p s u p p o r t f o r t h e p h y l o g e n e t i c a s s o c i a t i o n o fC r a s s u l a c e a e a n d P o d o s t e m a c e a e ( 8 % ) d o e s n o t i n s p i r e m u c h c o n f i d e n c e i n t h e d e g r e eo f i n t e r n a l s u p p o r t p r o v i d e d b y t h e rbcL da t a . We do , how ever , admi t t ha t t hep h y l o g e n e t i c as s o c ia t io n o f P o d o s t e m a c e a e , C r a s s u la c e a e , a n d H a l o r a g a c e a e r e p r e s e n ts amo re r easonab l e hyp o t hes i s t han t he pos i t ion o f t he o rde r a s a basa l ang i osp e rm l i neage .D esp i t e t he many morpho l og i ca l pecu l i a r i t i e s o f r i ve r -w eeds , t hey a r e unques t i onab l yd i co t y l edonous , and posses s s eve ra l s i mi l a r i t i e s t o bo t h C ras su l aceae and H a l o ragaceae .A l l t h r ee g roups have t e t r a sporang i a te , d i t heca l an t he r s , b i nuc l ea t e t r i co lpa t e po l l en , andd i s t i nc t s t y l e s . B o t h C ras su l aceae and Podos t emaceae have many ana t ropous , b i t egmi covu l es : s ep t i c i da l capsu l e s occur i n bo t h g roups . A qua t i c spec i e s a r e f ound i n a l l t h r eef ami l i e s , bu t t h i s may be supe r f l uous i f Wi l l i s ( 1915) w as co r r ec t i n conc l ud i ng t ha t t hef ami l y evo l ved f rom t e r r e s t r i a l p l an t s . I n t e r e s t i ng l y , many o f t hese f ea t u r e s a r e a l sos h a r e d w i t h H y d r o s t a c h y a c e a e , w h i c h d i f f e r f r o m C r a s s u l a c e a e a n d P o d o s t e m a c e a ees sen t i a l l y by t he i r un i t egmi c ovu l es and pa r i e t a l p l acen t a t i on . Maur i t zon (1933) a rgueds t ro n g l y t h a t e m b r y o l o g i c a l f e a tu r e s i n d i c a te d t h e r e d u c t io n o f H y d r o s t a c h y a c e a e a ndP o d o s t e m a c e a e f r o m t h e C r a s s u la c e a e . T h e r e s u lt s o f t h e rbcL ana l ys i s a r e i n compl e t ea g r e e m e n t w i t h t h i s s c e n a r i o . H o w e v e r , S c o g i n ( 1 9 9 2 ) c o m p a r e d t h e p h y t o c h e m i c a lp r o f il e s o f H y d r o s t a c h y a c e a e an d P o d o s t e m a c e a e , b u t f o u n d n o e v i d e n c e o f a c l o s er e l a t ionsh i p be t w e en t he f ami l ie s . H em pe l e t al . ( 1995) em phas i ze d t he un i t egmi c ovu l eso f H y d r o s t a c h y a c e a e t o s u g g e s t i ts p h y l o g e n e t i c p o s i ti o n s o m e w h e r e a m o n g t h e s u b c la s sA s t e r i dae . V erdc our t ( 1986 , p . 1 ) sugges t ed t ha t ev i dence con t r a ry to t he r e la t i onsh i p o fH y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e h a d b e c o m e " . . . t o o e x t e n s iv e to b e a r g u e da g a i n s t " .

H o w e v e r , m a n y t a x o n o m i s t s w h o h a v e d o u b t e d t h e r e l a t i o n s h i p o f P o d o s t e m a c e a ea n d H y d r o s t a c h y a c e a e h a v e g e n e r a l ly c o n c l u d e d t h a t t h e l a t te r f a m i ly s h o u l d b e p l a c e dphy l ogen e t i ca l l y nea r t he Sc rophu l a r i aceae , C a l l i tr i chaceae , o r H i ppur i daceae . C l ea r l yth e rbcL da t a p rov i de no ev i dence o f such an a s soc i a t i on . Thus w e a r e l e f t t o cons i de rt he t en t a t i ve hypo t hes i s o f f e r ed by t he cu r r en t rbcL ana l ys i s , and con t i nue t o s ea r ch fo rbe t t e r ev i dence fo r an a l t e rna t i ve p l acemen t o f t hese f ami l i e s . The re i s no doub t t ha tH y d r o s t a c h y a c e a e a n d P o d o s t e m a c e a e d i f f e r i n a n u m b e r o f f e a tu r e s ( R a u h a n d J ~ ig er-ZO rn , 1967) ; how ever , t he i r l eve l o f mol ecu l a r d i ve rgence , a s i nd i ca t ed by t he rbcLana l ys i s , does no t make t h i s an unexpec t ed ou t come , Thus , s t ud i e s t ha t have demon-s t ra t ed va r i ous d i f f e r ences be t w een t he g roups do no t neces sa r i l y p r ec l ude t he pos s i b i li t yof the i r re la t ionship .

A l t h o u g h a d e f i n i t i v e c o n c l u s i o n o n t h e p h y l o g e n e t i c p o s i t i o n o f e i t h e r t h e P o -d o s t e m a c e a e o r H y d r o s t a c h y a c e a e c a n n o t b e a c h i e v e d , th e a n a l y s is o f rbcL datar ep resen t s an i mpor t an t i n i t i a l s t ep i n de t e rmi n i ng t he co r r ec t r e l a t i onsh i ps o f t hesef ami l i e s . B ecause no o t he r comprehens i ve c l ad i s t i c ana l ys i s o f ang i ospe rms has ye t been


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24 D .H . Le s e t al . / A q u a t i c B o t a n y 5 7 (1 9 97 ) 5 - 2 7

conduc ted tha t inc ludes Podos temaceae , p rev ious hypo theses o f re la t ionsh ips s implyhave re f l ec ted the op in ion o f how va r ious au thors have pe rce ived the bes t 'ma tch ' o ffea tu res in th i s g roup wi th those o f o the r f lower ing p lan t fami l i e s. The app l i cat ion o fphy log ene t i c me thods in th i s s tud y p resen t s a hy po thes i s tha t can s e rve a s a reasonab lefo rum fo r fu r the r t e s t ing and s c ru t iny . The obse rva t ion tha t our re su l t s a re compa t ib lew i t h t h e o p i n i o n s o f m a n y c o n t e m p o r a r y t a x o n o m i s t s e n h a n c e s t h e r o b u s t n e s s o f t h epresen t hypo thes i s o f re la t ionsh ips fo r f ive r -weeds . S tud ie s a t h ighe r t axonomic l eve l sa re s e ldom l ike ly to y ie ld de f in i t ive ev idence o f re la t ionsh ips . The h igh l eve l o fh o m o p l a s y i n rbcL da ta i s ev idenced by the low cons i s t ency ind ices (< 35%) in a l lana lyses . However , the ove ra l l congruence o f the molecu la r da ta wi th the topo log ie sp resen ted was re spec tab le a s ind ica ted by re ten t ion ind ices o f 60 -64 % . Thus , the degreeof conf idence rende red by these re su l t s may no t be pa r t i cu la r ly h igh , bu t i s a rguab lyh i g h e r t h a n f o r c o n c l u s io n s r e a c h e d s i m p l y b y n o n e m p i f i c a l m e a n s .

5 . C o n c l u s i o n sThe rbcL sequences o f P odos tem aceae gene ra a re subs tan t ia l ly d ive rgen t f rom those

of o the r ang iospe rm s , inc lud ing the Hyd ros tachy aceae and o the r fami l i e s be l i eved torepresen t the c loses t ex tan t s is t e r g roup o f f ive r -weeds . The long re su l t ing b ranch , tha tsuppor t s Podos temaceae in pa rs imony ana lyses o f rbcL da ta , c rea te s p rob lems whena t t empt ing to roo t the s equences o f the comple te da ta s e t us ing d i s t an t non- f lower ingp lan t ou tg roups . A basa l pos i t ion o f Podos temac eae in the ang iospe rm s i s re so lved whennon - f lowef in g p lan t ou tg roup sequences a re used . A l tho ugh th i s re su l t supe r f i c ia l lysuppor t s c l a ims tha t Podos temaceae dese rve t axonomic rank equ iva len t to monoco ty le -dons and d ico ty ledons (Cusse t and Cusse t , 1988b,c) , we v iew th i s a s soc ia t ion a s aninc redu lous a r t i fac t o f long b ranch a t t rac t ion .

By re s t r i c t ing s equence compar i sons to ' eud ico t s ' (where the h ighes t p robab i l i ty o fre la t ionsh ip was expec ted) , a c l ade was re so lved tha t inc luded the Cras su laceae andHaloragace ae a s the s i s te r g roups to Hyd ros tachy aceae and Podos tem aceae . The mo no-ph y ly o f th i s c l ade was n o t s t rong ly suppor ted by boo t s t rap ana lys i s , bu t w as cons i s t en twi th the t axonomic op in ion o f s eve ra l con tempora ry sys tema t i s t s . The phy logene t i ca l l iance o f Podos tem aceae wi th the Hyd ros tachyac eae , Cras su laceae and Ha lo ragaceae ,i s p re sen ted a s a spec i fi c hypo thes i s fo r fu r the r eva lua t ion .

The mo rpho log ica l pecu li a r it i e s o f Podo s temaceae a re m i r ro red by ex tens ive mo lecu-la r d ive rgence , wh ich suppor t s the fami ly a s a mo noph y le t i c g roup dese rv ing o ftaxonomic recogn i t ion . A h igh degree o f molecu la r d ive rgence be tween Tristicha an do the r gene ra o f Podos temaceae suppor t s the recogn i t ion o f T r i s t i chaceae a s a s epa ra tef a m i l y .

A c k n o w l e d g e m e n t sW e t h a n k M . C l e l a n d a n d R . A a k j a r fo r t e ch n i c al s k il ls a n d K . J u n k o M i y a m o t o fo r

p rov id ing us wi th ma te r i a l o f Podos temaceae f rom Japan . Th i s work was pa r t i a l ly


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D.H. Les et al. /Aquatic Botany 57 (1997) 5-27 25

supported by NSF grants BSR 8817992 to DHL, DEB 9496053 to CTP, DEB-9629767to CTP and DHL and funding from The Intercambio Academico Office at the NationalUniversity of Mexico and Consejo Nacional de Ciencia y Tecnologia to ANR.

Ref erence s

A r b e r , A . , 1 9 2 0. W a t e r P l a n ts : A S t u d y o f A q u a t i c A n g i o s p e r m s . J . C r a m e r , N e w Y o r k , 4 3 6 p p .B e n t h a m , G . a n d H o o k e r , J .D . , 1 8 80 . G e n e r a P l a n ta r u m , V o l . 3 . W i l l ia m s a n d N o r g a t e , L o n d o n , p p . 1 0 5 - 1 1 5 .B h o j w a n i , S . S . a n d B h a t n a g a r , S . P ., 1 9 79 . T h e E m b r y o l o g y o f A n g i o s p e r m s , 3 r d e d n . V i k a s , N e w D e l h i, 2 8 0

P p .Burkhard t , G . , Sch i l d , W. , Becker , H . and Gruber t , M. , 1992 , B i pheny l s and xan t hones f rom t he Po -d o s t e m a c e a e . P h y t o c h e m i s t r y , 3 1: 5 4 3 - 4 5 8 .

Chase , M.W . e t a l ., 1993 . Phy l o gene t i cs o f seed p l an t s : an ana l ys i s o f nuc l eo t i de seque nces f rom t he p l as t i dg e n e rbcL. A n n . M i s s o u r i B o t . G a r d . , 8 0: 5 2 8 - 5 8 0 .C o o k , C . D . K . , 1 9 9 0 . A q u a t i c P l a n t B o o k . S P B A c a d e m i c P u b l i s h i n g , T h e H a g u e , T h e N e t h e r l a n d s , 2 2 8 p pC o r n e r , E . J .H . , 1 97 6 . T h e S e e d s o f D i c o t y l e d o n s , V o l . 1 . C a m b r i d g e U n i v e r s i t y P r e s s , C a m b r i d g e , U K , 3 1 1

Pp.Cronqu~s t , A . , 1981 . An In t eg ra t ed Sys t em o f C l ass i f i ca t i on o f F l ower i ng P l an t s . Co l umbi a Un i ver s i t y P ress ,New York , 1262 pp .

Cusse t , C . and Cusse t , G . , 1988a . E t ud e su r l es Pod os t em al es 9 . D~l i mi t a t ions t ax i nom i ques dans l esTr i s t i chaceae . Adanson i a , 10 : 149 -177 .

Cusse t , C . and Cusse t , G . , 1988b. E t ude su r l es Pod os t em al es . 10 S t ruc t u res fl o r a l es e t v6g&at i ves desT n s t i c h a c e a e . A d a n s o n i a , 1 0 : 1 7 9 - 2 1 8 .

Cusser , C . and Cusse t , G . , 1988c . E t ude su r l es Podos t em ops i da . 1 I . R~par t i t ion e t 6vo l u t ion des Tr i s t i chaceae .A d a n s o n i a , 1 0 : 2 2 3 - 2 6 2 .

Dah l g ren , R .M.T. , 1980 . A r ev i se d sys t em o f c l ass i f i cau on o f t he ang i ospe rms . Bo t . J. L i nn . Soc . , 80 91 - 124Dah l g ren , G . , 1989 . An updat ed ang i osp erm c l ass i f ica t i on . J L i nn . Soc . Bo t . , 100 : 197 -203 .Dorm er , K . J . , 1972 . Sho o t Organ i za t i on i n Vas cu l ar P l an t s . Sy rac use Un i ve r s i t y P ress , Sy racuse , 240 pp .E i c h l e r , A , W . , 1 8 86 . S y l l a b u s d e r V o r l e s u n g e n i i b e r P h a n e r o g a m e n k u n d e , 4 t h e d n . G . B o r n t r e a g e r , B e r l i n , 68

pp .E n d l i c h e r , S .L . , 1 83 7. G e n e r a P l a n t a r u m S e c u n d u m O r d i n e s N a t u r a l e s D i s p o s i ta , P a r t 4 , F r e i d n c h B e c k , W i e n ,

p p . 2 6 8 - 2 7 0 .E n d l i c h e r , S . L . , 1 8 3 9 . G e n e r a P l a n t a r u m S e c u n d u m O r d i n e s N a t u r a l e s D i s p o s i t a , S u p p l e m e n t 1 . F r e i d n c h

Beck , Wi en , p . 1375 .Eng l er , A . , 1930, Podos t em onal e s . I n : A . Eng l er and K . P ran t l (Ed i t o r s ) , D i e Nat i i r l ichen Pf l anzen fam i l i en ,

V o l . 1 8a . W . E n g e l m a n n , L e i p z i g , p p . 1 - 6 8 .F a h n , A . , 1 9 8 2 P l a n t A n a t o m y , 3 r d e d n . P e r g a m o n P r e ss , N e w Y o r k , 5 4 4 p pF e l s e n s t e i n , J . , 1 9 7 8. C a s e s i n w h i c h p a r s i m o n y a n d c o m p a t i b i li t y m e t h o d s w i l l b e p o s i t i v e l y m i s l e a d i n g S y s t .

Z o o l . , 2 7 : 4 0 1 - 4 1 0 .Fe l sen s t e i n , J . , 1985 . Conf i de nce l i mi t s on phy l ogen i es : an app roa ch us i ng t he boo t s t r ap , Evo l u t i on , 39 '

7 8 3 - 7 9 1 .Gardn er , G . , 1847. O bserva t i ons on t he s t ruc t u re and a f f i n i t i es o f t he p l an t s be l ong i n g t o t he na t u ra l o rder

P o d o s t e m a c e a e , t o g e t h e r w i t h a m o n o g r a p h o f t h e I n d ia n s p e c i e s . C a l c u tt a J . N a t . H i s t. , 7 : 1 6 5 - 1 8 9 .G r a h a m , S . A . a n d W o o d J r , C . E . , 1 97 5. T h e P o d o s t e m a c e a e i n t h e s o u t h e a s t e rn U n i t e d S t a t e s. J A r n o l d A r b . ,

5 6 : 4 5 6 - 4 6 5 .H e m p e l , A . L . , R e e v e s , P .A . , O l m s t e a d , R .O . a n d J a n s e n , R . K , 1 9 95 . Im p h c a t i o n s o f rbcL s e q u e n c e d a t a f o r

h i g h e r o r d e r r e l a t i o n s h i p s o f t h e L o a s a c e a e a n d t h e a n o m a l o u s a q u a t i c p l a n t Hydrostachys ( H y d r o -s t achyaceae) . P I . Sys t . Evo l . 194 : 25 -37 .Hen dy , M.D . and Pe nny , D . , 1989. A f r am ew ork fo r the quan t i t a t i ve s tudy o f evo l u t i onary t rees . Sys t . Zoo l . ,3 8 : 2 9 7 - 3 0 9 ,


22/23

26 D .H . Le s e t a L / A q u a t i c B o t a n y 5 7 (1 9 97 ) 5 - 2 7Hum bold t , F .W.H.A. von , Bonpland , A .J.A . and Kunth , C.S., 1816. Nova gene ra e t spec ie s p lan ta rum , Vol . 1

(fol. ed.). Lutetiae, Paris, p. 197.Hu tchinso n, J . , 1926. Th e Fam ilies of Flow ering Plants , Vol. 1 , Dicotyledons. M acm illan, London, 3 26 pp.J~iger-Ziirn, I . , 1992. Morp holo gie der Pod ostem acea e I I. lndotrist icha ramosissima ( Wi g h t ) V a n Ro y e n

(Tristichoideae). Fra nz Ste ine r Verlag, S tuttgart, 48 pp.J~iger-Ziirn, I . , 1995. M orph olog ie der Pod ostem acea e I II , Dalze l l ia cey lamca (Gard.) W ight (Tr ist ichoideae) .

Fran z Ste ine r Ver lag, Stuttgar t , 7 7 pp.Kapil , R.N., 1970. Podostemaceae . Bull . Indian Nat. Sci . Acad., 41: 10 4-10 9.Les , D .H., Ga rv in , D .K. and W impee , C.F., 1991 . Mo lecu la r evo lu t iona ry h is to ry o f anc ien t aqua t ic

ang iospe rms . Proc. Na t l Acad . Sc i. , USA , 88 :10 11 9-1 01 23 .Les , D .H., Ga rv in , D .K. and W impee , C.F., 1993 . Phy logen e t ic s tud ie s in the monoco t subc la ss Al i sma t idae :

Evid enc e for a reapp raisa l of the aquatic o rder Najadales. Molec . Phylog. Evol. , 2: 304 -31 4.Les, D.H. a nd Haynes, R.R., 1995. Syste ma tics of subclass Alisma tidae: a synthe sis of approaches. In: P.J .

Rudall , P. Cr ibb, D.F. Cu tler and C.J. Hum phries (Editors) , M onoc otyled ons: System atics and Evolution.Ro y a l Bo t a n i c G a r de n s , K e w , p p . 3 5 3 - 3 7 7 .

L indley , J ., 1830 . An In t roduc t ion to the Na tura l Sys tem of Botany . Longm an, Rees , Orme , B rown and G reen ,London , 374 pp .M addison , D .R. , 1991 . The d iscove ry and im por tance o f mul t ip le i s lands o f mos t -pa r s imonious t ree s . Sys t .Zool . , 40 : 315-328 .

M aheshw ar i , P . , 1945. The p lace o f ang iopse rm emb ryology in r e sea rch and teach ing . J . Ind ian Bot. Soc ., 24 :2 5 - 4 1 .

Ma ur i tzon , J. , 1933 . Uber d ie sys tema t i sche Ste l lung de r Fam i l ien Hydros tachyac eae und Podos temaceae . Bot .Not. , 1933: 9: 172-180.

M organ , D.R., Solt is, D.E. an d Rober tson , K.R., 1994. Systematic and evolution ary implica t ions of r b c Lsequ ence var ia t ion in Rosaceae . Amer. J . Bot. , 81: 89 0-9 03 .

Olmstead , R.G. and Pa lm er , J.D ., 1994. Chlorop la s t DNA systema tic s: a r ev iew of m e thods and da ta ana lys is .Amer. J . Bot. , 81: 1205-1224.

Presl, K.B., 1830. Reliq uiae Haenk eana e, Vol. I. J .G. Calve , Praha , 356 pp.Qiu , Y .L ., Chase , M .W., Les , D .H. and Pa rks , C.R. , 1993. Molecu la r phy logene t ic s o f the Magnol i idae :

Clad is t ic ana lyes o f nuc leo t ide sequences o f the p la s t id gene r b c L . An n. Missou r i Bot. Gard., 80:5 8 7 - 6 0 6 .

Rauh , W . and J~iger -Z i im, I ., 1967. Le p rob lbme de la pos i t ion sys t rma t ique des H ydros tachyac re s .A d a n s o n i a , 6: 5 1 5 - 5 2 3 .

Rom o Contreras, V ., Scogin, R., Philbr ick, C.T. an d No velo R.A., 1993. A phyto chem ical s tudy of se lec tedPodos tem aceae : sys tema t ic impl ica t ions . A li so , 13 : 513 -520 .

Rom bach , S . , 1911. Die Entwick lung de r Sam enknospe be i den Crassu laceen. Rec . Trav . Bot. N re r l . , 8 :1 8 2 - 2 0 0 .

Rut ishause r, R. , 199 5 . Dev e lopm enta l pa t te rns o f leaves in Podos temaceae com pared wi th m ore typica lf lower ing p lan ts : sa l ta t iona l evo lu t ion and fuzzy m orphology . Canad . J . Bot ., 73 : 1305-131 7 .

Rutishauser, R., 1997. Structura l and dev elopm enta l diversi ty in Podoste ma ceae ( river-weeds). Aquat . Bot. ,5 7 : 2 5 - 7 1 .Rut i shanse r , R. and Hub er K .A., 19 95 . The deve lopm enta l morphology of lndotrist icha ramosissima

(Podostemace ae-Tristichoideae). P1. Syst. Evol., 178: 19 5-2 23 .Sch le iden , M.J. , 1839. Sur la fo rma t ion de l 'ovu le e t l ' o r ig ine de l ' em bryo n de la Phane rogames . Ann . Sc i .

Nat. , Bot. , 11: 129-141.Schultz , C.H., 1832. NatiM iches System des Pf lanzen reichs. Au gust Hirschwald, Be r l in , 586 pp.Scogin , R. , 1992. Phy tochem ica l p rof i le o f Hydros tachys ins ign is (Hydrostachyaceae) . Aliso, 13: 47 1-4 74 .Sculthorpe , C.D., 1967. The Bio logy of Aq uatic Vascu lar Plants . E dwa rd Arn old, Lon don, 610 pp.Solt is , P.S. , Solt is , D.E. an d Doyle , J .J . (Editors) , 1992. Molec ular System atics of Plants . C hapm an and Hall ,

New York , 434 pp .Solt is , D.E., M orga n, D.R., G rable , A., Soltis , P.S. an d Kuzoff , R., 1993. M olecu lar system atics ofSaxifragaceae sensu stric to. Am er. J. Bot. , 80: 105 6-10 81.Swof ford, D .L ., 1993 . PAU P: Ph ylogene t ic Ana lys i s Us ing Pa r s imony , V ers ion 3 .1 .1. I l l ino is Na tura l H is to rySurvey, Cham paign , 162 pp.


23/23

D . H L e s e t a l . / A q u a t t c B o t an y 5 7 ( 1 9 9 7 ) 5 2 7 27

Takhtajan, A.L., 1980. Outline of the classification of flowering plants (Magnoliophyta) Bot. Rev, 46225-359.

Thorne, R F., 1992. Classlficatton and geography of the flowering plants. Bot. Rev., 58: 225-348.Tulasne, L.R., 1849 Podostemacearum Synopsis Monographica . Ann. Scl. Nat., Bot., 11: 87-114.Van Royen. P., 1951 The Podos temaceae of the New World. I Meded. Bot Mus. Utrecht, 107:1 151.Van Steems. C.G G.J., 1981. Rheophytes of the World. Sij thoff and Noordhoff, Rockville, Maryland, 408 pp.Verdcourt. B.. 198 6 Hydrostachyaceae In: R.M. Polhill (Editor), Flora of Tropical East Africa, no 135 A A

Balkema. Rotterdam, 6 ppWarming, E. 1888 Familien Podostemaceae Alhz,ndling III Danske Vidensk. Selsk Skr Nat. Math. 4

443-514Weddell, H.A., 1873. Podostemaceae. In: A.P. de Candolle et al (Editors), Prodromus Sy,~tematls Naturahs

Regni Vegetebalis, Vol. 17. Treuttel et Wtirtz, Pans , pp. 39- 89Went. F.A F C, 1910. Untersuchungen ueber Podostemaceen. Verh. Akad. Wet Amsterdam, 16 1-88. plates

1-15Willis, J .C , 1902. On the dorsiventrality of the Podostemaceae w~th reference to current ~lew's on their

evolution Ann Bot, 16:593-594Wdlis, J.C , 1915 A new natural famdy of flowering plants--Tn~,tlchaceae. J Linn. Soc. Bot., 43: 40 -5 4Willis, J (". 1926. The evolution of the Tnstlchaceae and Podostemaccae Ann Bot., 40 349 367

the phylogenetic position of river-weeds podostemaceae insights from rbcl sequence data

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