1083 a Ground-Breaking Study Published 5 Years Ago Revealed That

Home , Bdallophytum, Cuscuta, Mitrastemon, Myrtales, Rafflesiaceae

American Journal of Botany 100(6): 1083–1094. 2013.

SPECIAL INVITED PAPER—EVOLUTION OF PLANT MATING SYSTEMS

P OLLINATION AND MATING SYSTEMS OF APODANTHACEAE AND THE DISTRIBUTION OF REPRODUCTIVE TRAITS IN PARASITIC ANGIOSPERMS 1

S IDONIE B ELLOT 2 AND S USANNE S. RENNER 2

Systematic Botany and Mycology, University of Munich (LMU), Menzinger Str. 67 80638 Munich, Germany

• Premise of the study: The most recent reviews of the reproductive biology and sexual systems of parasitic angiosperms were published 17 yr ago and reported that dioecy might be associated with parasitism. We use current knowledge on parasitic lineages and their sister groups, and data on the reproductive biology and sexual systems of Apodanthaceae, to readdress the question of possible trends in the reproductive biology of parasitic angiosperms. • Methods: Fieldwork in Zimbabwe and Iran produced data on the pollinators and sexual morph frequencies in two species of Apodanthaceae. Data on pollinators, dispersers, and sexual systems in parasites and their sister groups were compiled from the literature. • Key results: With the possible exception of some Viscaceae, most of the ca. 4500 parasitic angiosperms are animal-pollinated, and ca. 10% of parasites are dioecious, but the gain and loss of dioecy across angiosperms is too poorly known to infer a statisti- cal correlation. The studied Apodanthaceae are dioecious and pollinated by nectar- or pollen-foraging Calliphoridae and other ﬂ ies. • Conclusions: Sister group comparisons so far do not reveal any reproductive traits that evolved (or were lost) concomitant with a parasitic life style, but the lack of wind pollination suggests that this pollen vector may be maladaptive in parasites, perhaps because of host foliage or ﬂ owers borne close to the ground.

Key words: Apodanthaceae; dioecy; ﬁ eld observations; ﬂ y pollination; phylogeny; sexual system.

A ground-breaking study published 5 years ago revealed that of Barkman et al., remains unclear. The single genus of this among fl owering plants, parasitism evolved at least 13 times family has been placed in Myrtales, Rosales, Santalales, Saxi- (Barkman et al., 2007), namely in the Apodanthaceae, Balano- fragales, and Sapindales (Nickrent et al., 2005; Jian et al., 2008; phoroaceae, Cassytha (Lauraceae), Cuscuta (Convolvulaceae), Qiu et al., 2010 ; Zhang et al., 2009 ), but the situation is not yet Cynomoriaceae, Cytinus (Cytinaceae), Hydnoraceae, Krameria settled ( Zhang et al., 2011 ). (Krameriaceae), Lennoaceae, Mitrastemon (Mitrastemonaceae), With the independent parasitic lineages of fl owering plants Raffl esiaceae, higher Santalales, and Orobanchaceae (exclud- now clear, it is timely to return to the question of traits that may ing the fi rst-branching genus Lindenbergia ; Fig. 1 ). Together, be associated with the evolution of parasitism. This question these clades may comprise some 4500 species, 2040 of them in was last addressed in 1995 in a review of the reproductive biol- the Orobanchaceae and another 2100 in the Santalales ( Table 1 ogy of parasitic angiosperms that found no discernible trends provides species numbers and references). The phylogenetic unique to parasitic plants ( Molau, 1995 ). The review followed placements of 12 of the 13 parasite lineages found by Barkman the classifi cation most accepted at the time (Cronquist, 1988), et al. (2007) have been upheld in studies with denser gene or which lumped several parasite groups since revealed to be inde- taxon sampling (Nickrent, 2007; Vidal-Russell and Nickrent, pendent, such as Apodanthaceae and Raffl esiaceae, and Bal- 2008a , b ; Filipowicz and Renner, 2010 ). Only the placement of anophoraceae and Cynomoriaceae, while separating others since Cynomoriaceae, which was unsupported in the preferred topology revealed to have a single ancestor, such as Eremolepidaceae and Santalaceae. The focus of the review also was not evolu- 1 Manuscript received 6 December 2012; Revision accepted 25 March tionary since relevant sister group relationships were not yet 2013. known. The authors thank J. Kuijt for discussion of parasite biology; D. Plowes, An obvious parallel evolutionary trend in parasitic angio- M. Coates Palgrave, and M. Hyde for help with fi eldwork in Zimbabwe; sperms is the reduction of the capacity to photosynthesize. The D. Plowes, B. Schlumpberger, and G. Gerlach for photos; S. Zarre and loss of photosynthetic capacity is complex because genes cod- Y. Salmaki for help with fi eldwork in Iran; M. Correa and C. Galdames for ing for photosynthesis are partly located in the chloroplast ge- information on species from Panama; K. Thiele for information from Australia; nome, partly in the nucleus, and plastids are involved in other G. Ceccantini for information from Brazil, B. Muller, T. Dikow, S. Marshall, metabolic pathways than those directly related to photosynthe- T. Pape, K. Rognes, E. Kameneva, D. Doczkal, and M. Kotrba for identifi cations of fl ies; M. Sharaf and B. Taylor for ant identifi cations; and D. Plowes and an sis ( Neuhaus and Emes, 2000 ; Krause, 2012 ). All holoparasites anonymous reviewer for pointing us to important literature. investigated so far probably therefore still have a plastid ge- 2 Authors for correspondence (e-mails: [email protected], [email protected]) nome ( Table 1 : column 4). Other life history or morphological traits that could have undergone parallel evolution in parasites doi:10.3732/ajb.1200627 concern the timing of reproduction (especially in seasonal

Fig. 1. Phylogenetic relationships, sexual systems, and main pollinators of the 13 parasitic angiosperm lineages and their sister groups. Autotrophic lineages are shown in green, parasitic ones in black. aD = androdioecy, D = dioecy, H = hermaphrodism, M = monoecy, pM = polygamomonoecy. Question marks indicate uncertainty. See Table 1 for references. climates), the mode of seed dispersal ( Kuijt, 1969 ), or conceiv- dioecious species, only 1% contained parasitic species (Renner ably also the mode of pollination. For example, animal pollina- and Ricklefs, 1995 ). A seeming correlation between dioecy and tion might be more reliable than wind pollination if parasite a parasitic life style was also noted in the fl ora of the southeast- fl owers are often located near the ground or under host foliage. ern United States: ca. 17% of Carolina heterotrophs and myco- Because most parasitic lineages can only be studied in natu- heterotrophs are dioecious ( Conn et al., 1980 ). More recently, ral conditions, not in the laboratory or botanical gardens, knowl- an analysis of diversifi cation rates in parasitic plants (Hardy edge of their reproductive biology is patchy. Pollinators of and Cook, 2012 ) revealed that a parasitic life style may contrib- Cynomoriaceae and Lennoaceae have never been observed, and ute to high extinction risks (for hosts and parasites alike), and the fi rst data on the pollination of Apodanthaceae are reported this risk might be compounded by a dioecious sexual system in in the present study. Parallel or convergent evolution in the which species may suffer a competitive disadvantage because reproductive modes of parasitic angiosperms is thus an open only half of the individuals in a dioecious population are seed question. In terms of sexual systems, a study published 17 yr bearing (Heilbuth et al., 2001; Vamosi and Vamosi, 2004). It ago suggested an overrepresentation of dioecy among parasitic would therefore be surprising if dioecy were indeed overrepre- angiosperm genera. This was based on the fi nding that of 959 sented among parasitic angiosperms. angiosperm genera with dioecious species, at least 43 (4%) also Here we use today’s data on the placement of the 13 parasite contained parasitic species. Of the angiosperm genera lacking families and their reproductive biology to readdress the question June 2013] BELLOT AND RENNER—REPRODUCTIVE BIOLOGY OF PARASITIC ANGIOSPERMS 1085

3 44 31

7 58 26

21 ies ies foraging bees abiotic foraging short- tongued insects ﬂ maybe sometimes abiotic animal- pollinated foraging birds, moths, bees, wasps, ﬂ foraging animals sister group Pollination in Biotic or Nectar- Insects Insects, Mostly Nectar- Nectar- Typically Typically

7 Bees

20 25, 7

7 31 34 2

8 Population monoecious monoecious or dioecious (or other sexual systems) hermaphrodite, some dioecious or dioecious monoecious or dioecious or dioecious monoecious or dioecious sexual sexual system in sister group Mostly Hermaphrodite, Mostly Hermaphrodite Hermaphrodite, Hermaphrodite Hermaphrodite,

2 8 7 31 34 Hermaphrodite,

15 7 Hermaphrodite Hermaphrodite 20 25, 7 Flower Flower unisexual unisexual unisexual unisexual bisexual also but unisexual bisexual bisexual bisexual bisexual unisexual unisexual sister group phenotype in Mostly ?? ? Bisexual or Bisexual Mostly Bisexual Hermaphrodite Nectar- Unisexual or Unisexual Bisexual Bisexual Bisexual Mostly or Bisexual or Bisexual

33 52

24 33

57 57

57 no. of species no. of genera/ Sister group and or some) up to 2600 species in 109 genera Cryptocaryeae ca. 40/1730 Convolvulaceae lineage? Balsaminaceae, and Marcgraviaceae, Tetrameristaceae 106–138/1552– 3304 parasites + 11–13/44–83 autotrophs (Orobanchaceae) 3-30 22/285 10275 species 3/3 5–8/480 8/170 Ehretiaceae Lindenbergia Somewhere in Somewhere

Lauraceae without Lauraceae Saxifragales Some Muntingiaceae Aristolochiaceae Zygophyllaceae excluding Ericales

14 17 18 23 27 58 27, 53 12 42, 43 and annual annual and annual Perennial Cucurbitales (all Perennial Unknown Other Santalales Mostly Perennial Perennial Perennial

Perennial Perennial 58 27 58

;

Perennial 6

Perennial 48 ; 19; 62, 63 32 58 22

; 67 50

12 51, 58 dispersal Longevity other animals eating small fruits animals beetles rain wash rain wash and ants with spines mammal water water mechanical rain Fruit or seed Birds or Fruit-eating Wind, rain Wind, Birds, rain Rodents, Birds Rodents Mammals Nuts barbed Grazing Possibly rain

or 65

; 22

42 6, 65 22, 64 11 ies ower ower ; bats 66 27 58 ; 18 ies or bees, small owers, study) fl extrapolating from fl morphology fl amounts of nectar visited by nectar foragers morphology and scent, fl prob. bees Lepidoptera bees beetles bees shrews Zosterops ants, moths, beetles on pollen? Flies (this Shallow tubular tubular Shallow Nectar-foraging Nectar-foraging ,

; 61, 54 and 60

Epifagus Epifagus refl exa refl Pilostyles 49 investigated Pollination aethiopica hamiltonii P. (this study) ongoing: 7 other species Cuscuta C. exaltata, ora C. obtusifl C. gronovii virginiana Plastid genome Ongoing: None Flies, wasps, NoneNone Flies feeding Ants Published: None Published: Nectar-foraging ,

None Oil-collecting None None Probably Meliphagidae , Noneon Based 12 42 4 18 55 7 27 27 12 12

16 species or dioecious 5 or dioecious 21 dioecious polygamo- monoecious or dioecious 6 no. of dioecious Population sexual Population sexual system, Minimum Monoecious or Monoecious,

Monoecious 16 18 Hermaphrodite Hermaphrodite Hermaphrodite Hermaphrodite Hermaphrodite Hermaphrodite 4 Hermaphrodite 12 42 7 27 27 12 12 Flower Flower bisexual bisexual bisexual bisexual phenotype Unisexual Monoecious Bisexual Bisexual Unisexual Unisexual Bisexual Bisexual Bisexual Bisexual Bisexual Bisexual Unisexual and Unisexual

(Lauraceae):

43 1. Biological traits of parasitic clades and their sister analyzed in this review. 2/18 (= Mitrastemonaceae): (excluding Lindenbergia 96/2040 1/2 16/42 (Convolvulaceae): (Convolvulaceae): 1/200 1/21 (= Krameriaceae): 1/18 (= Cynomoriaceae): 1/1 ABLE Cytinaceae: 2/10 or Unisexual Apodanthaceae: Mitrastemon Orobanchaceae Balanophoraceae: Cuscuta Cassytha Krameria Lennoaceae 2/4 Bisexual T clades: Parasitic No. of genera/ No. of species Hydnoraceae: 2/9 Bisexual Cynomorium 1086 AMERICAN JOURNAL OF BOTANY [Vol. 100 Goto 5 Frost et al., sister group Pollination in 50 Mostly biotic de Figueiredo Encyclopædia 21 34 Yatskievych, G., ? 8 63

45 Hannon Williams and Pahlevani and Akhani, 28 Nickrent, 1997 onward . Nickrent, 1997 onward 41 12 Wicke, 2012 . 49 Population dioecious Govindappa and Shivamurthy, and Shivamurthy, Govindappa sexual sexual system in sister group 66 Nickrent, 2007 . Shumei and Murata, 2003 . 20 4 Monoecious or Monoecious 45 Yatskievych, 1985 . Hermaphrodite 8 62 Hardy and Cook, 2012 . Hardy and Cook, 2012 33 Flower Flower sister group Narbona et al., 2005 . Narbona et al., 2005 phenotype in Ecroyd, 1996 . Unisexual Unisexual Johnson et al., 2011 . 40 65 11 Kuijt, J., University of Victoria, British Columbia, Victoria, of J., University Kuijt, Bisexual Bisexual 56 8 Jacobsohn et al., 1987 . Jacobsohn et al., 1987 Jepson Flora Projects, 2012 . Jepson Flora Projects, 2012 48

27 38

57 McNeal et al., 2007 . McNeal et al., 2007 61 Nickrent, 2011 . no. of species no. of genera/ Sister group and Encyclopædia Britannica, 2012 . Britannica, 2012 Encyclopædia 218/5735 10 Yoder, Yoder, 1998 . Sakai, 2002 . 3 47 26 Bouman and Meijer, 1994 . 1994 Bouman and Meijer, Coulaceae 2–3/2–4 Pé labon et al., 2012 . Garcia-Franco and Rico-Gray, 1997 . 1997 Garcia-Franco and Rico-Gray, 32 39

19 4 Alvarado-Cárdenas, 2009 . 55 some annual in Thesium Wolfe et al., 1992 . Perennial, 60

10,56 Perennial? Euphorbiaceae 32 Davis et al., 2007 . et al., 2007 Davis 38 de Vega et al., 2009 . et al., 2009 Vega de Allen and Hiscock, 2008 . Allen and Hiscock, 2008 9 18 dispersal Longevity Schaefer and Renner, 2011 . 2011 Schaefer and Renner, explosion explosion 2 Nair and Narayanan, 1962 . Funk et al., 2007 . Funk et al., 2007 Fruit or seed Bennett and Mathews, 2006 . 2006 Bennett and Mathews, Mammals Birds, wind, 25 54 46

c Beaman et al., 1988 . Beaman et al., 1988 ;

59 68

y- 59 58, 68 Beehler, 1994 . ies ies 1 two two fl pollinated species of Arceuthobium also may receive wind-carried conspecifi pollen fl birds, moths, bees, wasps, fl García et al., 2004 . García et al., 2004 . 17 Nickrent et al., 2010 . Nickrent et al., 2010 8 Nickrent et al., 2002 . Nickrent et al., 2002 Banziger and Hansen, 2000 . Banziger and Hansen, 2000 24 37 Radcliffe-Smith, 2001 . 45 Yatskievych and Mason, 1986 . and Mason, 1986 Yatskievych Heide-Jorgensen, 2008 . investigated Pollination 53 58 Plastid genome None Ovipositing None Nectar-foraging Schlumpberger, B., Herrenhäuser Garden, Hannover, Germany, personal communication. Germany, B., Herrenhäuser Garden, Hannover, Schlumpberger, Materials and Methods Nickrent et al., 2005 . Nickrent et al., 2005 , 64 16 , 419 36, 37 8 Gottschling, M., et al., University of Munich, personal communication. Gottschling, M., et al., University 31 Watson and Dallwitz, 1992 . 1992 and Dallwitz, Watson Tennakoon et al., 2007 . et al., 2007 Tennakoon 7 Barcelona et al., 2011 . Barcelona et al., 2011 species 23 36 5–30 dioecious or trioecious monoecious or dioecious (or other sexual systems) Kubitzki and Kurz, 1984 . 1984 and Kurz, Kubitzki no. of dioecious Population sexual Population sexual system, Minimum Monoecious, Hermaphrodite, and Olmstead, 2004 . and Olmstead, 2004

44 ć Rohwer, 1993 .

15 Stevens, 2001 . onward 8 APG, 2003 . 36, 37 57 30 Player, 1979 . 68 Flower Flower Stefanovi unisexual unisexual unisexual unisexual phenotype 52 Bisexual or Bisexual Costea, 2007 . 43 Smets and Pyck, 2003 . Smets and Pyck, 2003 Wikipedia, 2013 . 35 14 Wang et al., 2009 . et al., 2009 Wang Maass and Musselman, 2004 . Maass and Musselman, 2004 Borchsenius and Olesen, 1990 . Borchsenius and Olesen, 1990 29 6 22

8 aceae, : for sources on species and genus numbers see de Vega et al., 2011 . et al., 2011 Vega de Wright, 2011 . Sakai et al., 1995 . Sakai et al., 1995 67 42 51 1. Continued. esiaceae: 3/34 or Bisexual Notes (Amphorogynaceae, Aptandraceae, Cervantesiaceae, Comandraceae, Loranthaceae, Misodendraceae, Nanodeaceae, Octoknemaceae, Olacaceae, Opiliaceae, Santalaceae, Schoepfi Thesiaceae, Viscaceae and Ximeniaceae, excluding Balanophoraceae and the three basal- branching lineages Coulaceae, Strombosiaceae, Erythropalaceae): 148/2111 Nelson, 2008 . ABLE et al., 2012 . et al., 2012 Santalales T clades: Parasitic No. of genera/ No. of species 13 et al., 2008 . et al., 2008 1975 . . Britannica, 2012 Finke, 2011 . 2011 Finke, Canada, personal communication. 2011 . 2003 . Missouri Botanical Garden, personal communication. Raffl June 2013] BELLOT AND RENNER—REPRODUCTIVE BIOLOGY OF PARASITIC ANGIOSPERMS 1087 whether parasitic angiosperm lineages exhibit shared traits in however, any strong trend, such as lack of wind pollination or their pollination biology, dispersal modes, or sexual systems. preponderance of bisexual fl owers, can be picked up even from We also describe the pollination and mating system of two spe- incomplete data. cies of Apodanthaceae. Apodanthaceae comprise 18 species in two genera, Apodanthes and Pilostyles (a third genus name, Berlinianche , has never been validly published). They occur in North and South America, Africa, Iran, and Australia. Like MATERIALS AND METHODS most other Cucurbitales, Apodanthaceae have unisexual fl owers. The fl owers and fruits measure 4–10 mm in diameter (Blarer Fieldwork on Pilostyles aethiopica and Pilostyles haussknechtii — Pilo- et al., 2004 ; present paper) and appear on the hosts’ stems and styles aethiopica Welwitsch was studied between 16 and 29 February 2012 in the Mukuvisi woodlands, at 17° 50 ′ 44.16 ″ S and 31 ° 05’24.12 ″ E in Harare, Zim- branches once a year (Fig. 2). Insect pollination had been in- babwe. These woodlands are protected. The parasite fl owers were borne on the ferred from fl ower morphology and color, and occasional ob- branches of 1.5 to 4 m tall trees of Julbernardia globifl ora (Benth.) Troupin servations of visitors in Africa, Australia, and Brazil ( Dell and (Fabaceae). Pilostyles haussknechtii Boissier was studied between 28 May and Burbidge, 1981; Blarer et al., 2004; Brasil, 2010). Blarer et al. 11 June 2012 in two populations in Iran, one north of Teheran in the province of Alborz (35° 55 ′ 14.09 ″ N and 51° 03 ′ 12.78 ″ E), the other in the province of (2004) detected no fl ower odor in the African Pilostyles (Berlin- ° ′ ″ ° ′ ″ ianche) aethiopica Welwitsch, while Blassingame (1968) per- Isfahan (32 55 11.3 N and 50 41 23.1 E). The local vegetation type was steppe (Fig. 2), and the hosts were 0.20 to 1 m tall shrubs of Astragalus fl occosus Bois- ceived an indoloid odor in the Californian P. thurberi Gray. sier near Teheran and of A. verus Olivier in Isfahan. Herbarium vouchers of the Questions about reproductive trait correlations across angio- parasites and their hosts have been deposited in the herbarium of Munich, M sperms are diffi cult to answer because of insuffi cient data; ( S. Bellot 5 to S. Bellot 56 ).

Fig. 2. Habits and fl oral morphology of two species of Pilostyles . (A) Pilostyles haussknechtii on Astragalus fl occosus near Isfahan, Iran. (B) Pilo- styles aethiopica on Julbernardia globifl ora in the Mukuvisi woodlands, Harare, Zimbabwe. (C) Possible monoecy, with male (left) and female (right) fl owers of P. haussknechtii on a stem of A. fl occosus . (D) Male fl ower of P. aethiopica . (E) Female fl ower of P. aethiopica (courtesy of D. Plowes). 1088 AMERICAN JOURNAL OF BOTANY [Vol. 100

At the Harare site, 20 host trees carrying numerous parasite fl owers were a fl owering host, fl owers of host and parasite never occurred on selected. Male Pilostyles aethiopica fl owers have a shiny, white hair crown at the same host stem. the top of their central column, whereas female fl owers do not (Fig. 2), making it possible to sex fl owers by eye. At the Isfahan site, 27 infected shrubs were studied, and at the Teheran site, 26 infected and 123 uninfected shrubs. To sex Flowers visitors and fl oral scent — At the Harare site, four the fl owers of P. haussknechtii required close observation or prying open fl ow- fl y species (Fig. 3A–D) and two ant species (Fig. 3E, F) were ers, and it was therefore only possible to sex a subset of the fl owers on each observed on both fl ower sexes and during more than one obser- host. At the Teheran site, we also studied whether host fl owering was more or vation period. The ant species Camponotus fl avomarginatus less abundant in infected vs. noninfected hosts. Mayr, 1862 and Lepisiota capensis Mayr, 1862 (both Formici- In Harare, parasite fl owers in fi ve host tree patches were observed during nae; vouchers S. Bellot 4 , 5 , 6 ) are unlikely pollinators because 10-min periods between 0900 and 1700 hours on several days for a total of 36 observation periods. Notes were taken on fl ower sex, scent, number and kind of they return to the nest between forays to different food sources. insect visitor, and visitor behavior. Visitors were also photographed, and cap- In Harare, the most common fl ies were Chrysomya chloropyga tured visitors have been deposited in the zoological collections of Munich (Zo- Wiedemann, 1818 (voucher S. Bellot 1 ; Fig. 3B ) and Sto- ologische Staatsammlung München; vouchers S. Bellot 1 to 6 ). Photographs morhina sp. (not captured; Fig. 3A), both belonging to the and specimens were studied and identifi ed by the fl y and ant specialists listed in Calliphoridae. In Iran, the fl y Timia (Empyelocera ) spec. aff. the acknowledgments. For the six most common visitors to both fl ower sexes, abstersa (Ulidiidae; voucher S. Bellot 7; Fig. 3G) was common visitation frequency was quantifi ed. The scent of male and female fl owers was assayed by letting it accumulate in closed small glass vials. Nectar presence on fl owers of both sexes. At both sites, fl ies alighted on branches was tested with diabetes testing strips that were inserted into fl owers. near fl owers (which were too small for them to land on), ap- proached the fl owers, and then probed the stamens or the base Assessment of reproductive traits in parasitic angiosperms and their sister of the fl oral column with their proboscis, apparently looking for groups —All angiosperm genera recorded as parasitic were databased; follow- pollen grains or liquid. Their entire heads, including the upper ing previous reviews, we included Santalales lineages thought to be parasitic part of the proboscis, came in contact with the anther ring or based on their phylogenetic position but lacking information on haustoria for- stigma. In Harare, fl oral scent could be perceived throughout mation. Most important sources were D. L. Nickrent’s Parasitic Plant Connec- tion website (http://www.parasiticplants.siu.edu/UsingPPC.html, last accessed the day, with no apparent change in intensity; female fl owers on 28 October 2012) and R. Walker’s Parasitic plants database (http://www. produced scent before they opened. In Iran, fl ower scent was omnisterra. com/bot/pp_home.cgi, last accessed 28 October 2012). For each not always perceptible and was weak, even after accumulation genus, species names were downloaded from Tropicos (http://www.tropicos. in glass vials (done at ca. 1000 and 1500 hours). No difference org), and synonyms or illegitimate names were deleted. The Plant List (http:// in scent between male and female fl owers could be perceived in www.theplantlist.org/) and the Angiosperm Phylogeny Website (Stevens, either species. Nectar presence could not be verifi ed with the 2001 ) were also consulted. This resulted in a table of 5058 accepted species names (Appendix S1; see Supplemental Data with the online version of this diabetes testing strips because nectar quantities were too min- article); for the largest groups, Cuscuta, Orobanchaceae, and Santalales, we ute. No insect eggs or larvae were found in any fl ower. accepted species numbers of 200 (Digital Atlas of Cuscuta , Costea [2007] ), 2040 ( Stevens, 2001 ), and 2111 ( Nickrent et al., 2010 ), slightly higher (Cuscuta ) Reproductive traits of parasitic angiosperms and their sister or lower than those obtained from other sources. groups — Of a total of 4510 parasitic species, about 10% are Traits scored for parasitic species (Appendix S1) were fl ower morphology (whether unisexual or bisexual), population sexual system(s), longevity (an- dioecious (Table 1 and Appendix S1). Dioecy is found in Apo- nual, perennial), pollinator(s), and seed and fruit dispersal (partly assessed only danthaceae, Balanophoraceae, higher Santalales, Cytinaceae, and from fruit types). Data sources (Appendix S1) included online fl oras, websites, Raffl esiaceae, and in the sister groups of three of them ( Table 1 ). articles in journals, and books of parasitic plants, most importantly Kuijt (1969) Of the eight parasitic lineages lacking dioecious species, fi ve and Heide-Jorgensen (2008) . The scored data were used to construct a summary have sister groups that include dioecious species ( Table 1 ). table at the parasitic family level or other relevant taxonomic levels (Results). Table 1 also shows that all parasitic lineages contain perennial species, including trees (Fig. 4E), while fi ve contain perennial as well as annual species. Only Cuscuta is predominantly an- RESULTS nual. All dioecious parasite species appear to be perennial. With the exception of the Viscaceae genus Arceuthobium , in Sexual systems and fl owering of Apodanthaceae — Table 2 which nectar-offering and fl y-pollinated species may some- summarizes the results of the distribution of fl ower morphs in times receive wind-carried conspecifi c pollen (Player, 1979), the Zimbabwean and Iranian populations. In Zimbabwe, 12 hosts parasitic species are animal-pollinated, with all pollinator cate- carried only male parasite fl owers, seven only female fl owers, gories from vertebrates to insects represented ( Table 1 ). Figure 4 and one fl owers of both sexes. In the two Iranian populations, shows some of the pollination syndromes represented ( Fig. 4C : 19 or 17 host shrubs carried only male parasite fl owers, while bee/bumblebee pollination, Fig. 4D: animal pollination with six or 10 carried only female parasite fl owers. One shrub car- trapping). Among parasites’ sister groups, several contain wind- ried parasite fl owers of both sexes (Fig. 2C). At the Teheran site, of pollinated species (Table 1). Fruit or seed dispersal by animals 123 uninfected hosts almost all fl owered, while of the 26 infected predominates in the parasite lineages (Fig. 4A, B), except for hosts, only seven fl owered. When the parasite was fl owering on Cynomoriaceae, Lennoaceae, and some Orobanchaceae, the

T ABLE 2. Host distribution of male and female ﬂ owers of Pilostyles aethiopica and P. haussknechtii

Hosts with only male Hosts with only female Hosts with male and female Species Population Infected hosts parasite fl owers parasite fl owers parasite fl owers

P. aethiopica Mukuvisi woodlands 20 12 7 1 P. haussknechtii Teheran 26 19 6 1 Isfahan 27 17 10 0 June 2013] BELLOT AND RENNER—REPRODUCTIVE BIOLOGY OF PARASITIC ANGIOSPERMS 1089

Fig. 3. Flower visitors of (A–F) Pilostyles aethiopica in Zimbabwe and (G) P. haussknechtii in Iran. (A) Stomorhina sp. (Calliphoridae). (B) Chry- somya chloropyga (Calliphoridae). (C, D) Fly fl ower visitors. (E) Lepisiota capensis (Formicidae). (F) Camponotus fl avomarginatus (Formicidae). (G) Timia ( Empyelocera ) sp. aff. abstersa (Ulidiidae). The fl ower diameter is 4–6 mm. seeds of which are dispersed by rain splash or wind (cf. refer- system ( Zhang et al., 2006 ). A study of the Brazilian species ences in Table 1 ). P. ingae (Karsten) Hooker recently reported that 1.3% of the male fl owers had small pistils, but were functionally male (Brasil, 2010: The numbers of dissected fl owers were not given.). In the DISCUSSION species studied here, P. aethiopica and P. haussknechtii , dioecy appears to be the normal sexual system ( Table 1 ). Among the Sexual systems and reproductive biology of Apodanthaceae — remaining Apodanthaceae, three or four are monoecious, Flowers of Pilostyles aethiopica and P. haussknechtii were namely the Australian P. collina B. Dell and P. coccoidea strictly unisexual, without traces of aborted organs of the K.R.Thiele ( Thiele et al., 2008 ), the Brazilian Apodanthes mi- other sex. Unisexual fl owers are the ancestral condition in the narum Vattimo (de Vattimo, 1973), and possibly the Brazilian Cucurbitales, apparently combined with a dioecious mating P. stawiarskii de Vatt. ( de Vattimo, 1950 ). Most remaining species 1090 AMERICAN JOURNAL OF BOTANY [Vol. 100

Fig. 4. Examples of reproductive traits of parasitic angiosperms. (A) Seedlings of the hemiparasite Krameria sp. (Krameriaceae) at the foot of a host (Cactaceae) with barbed seeds (inset) adapted to exozoochory. (B) Bisexual fl owers and fruits of the bird-pollinated and dispersed Tristerix longibracteatus (Loranthaceae). (C) Herb habit of the root parasite Orobanche lavandulacea (Orobanchaceae) and close-up of its bee-pollinated infl orescence (inset). (D) Bisexual fl ower of the root holoparasite Prosopanche americana (Hydnoraceae) in male phase (courtesy of B. Schlumpberger). (E) Tree habit and infl orescence (inset) of the hemiparasite Gaiadendron (Loranthaceae). Images A–C, E: courtesy of G. Gerlach. June 2013] BELLOT AND RENNER—REPRODUCTIVE BIOLOGY OF PARASITIC ANGIOSPERMS 1091 are dioecious [ P. ingae and P. thurberi (Brasil, 2010); the Aus- proliferation seen in parasitic angiosperms ( Hardy and Cook, tralian P. hamiltonii Gardner (Thiele et al., 2008), the American 2012 ). However, one would then expect dioecious parasite lin- P. mexicana (Brandegee) Rose, P. blanchetii (Gardner) Brown, eages to be more species poor than the strictly bisexual ones, P. calliandrae (Gardner) Brown (de Vattimo, 1971), A. tribrac- which is not the case ( Table 1 ). The situation may be complicated teata Rusby ( Rusby, 1920 ), and A. caseariae Poiteau ( Poiteau, by additional correlations, for example, between host specifi city 1824)]. The sexual system of the American species P. galactiae and dioecy, presumably both requiring highly effi cient dispersal Ule, P. goyazensis Ule, and A. berteroi (Guill.) Gardner, and of mechanisms to generate the dense seed shadow required to main- the African P. holtzii Engler is unknown, the later species being tain viable populations ( Wilson and Harder, 2003 ). known only from a type destroyed during World War II. Dioecy is less likely in families of herbs and more likely in The endoparasitic lifestyle of Apodanthaceae makes it diffi cult families containing shrubs and trees (Renner and Ricklefs, to decide whether cases of monoecy in fact represent multiple in- 1995 ), and we therefore looked at the longevity of parasitic fections with two or more parasite individuals, each representing plants with or without dioecious species. All 13 parasitic lin- one sex. Next-generation sequencing data that will facilitate the eages contain perennial species, while four or fi ve also contain design of highly variable genetic markers have now been generated annual species ( Table 1 ). None of the annual species is dioe- for Pilostyles hamiltonii and P. aethiopica and may resolve this cious (the longevity of Balanophoraceae is still unknown), question (S. Bellot and S. S. Renner, unpublished data). Nothing is matching the fi nding of Renner and Ricklefs (1995). A peren- known about germination and initial haustorium formation, but nial life cycle may be advantageous for parasites, especially animal dispersal is certain because the fruits are small red, orange, when hosts are diffi cult to fi nd (i.e., when a huge seed shadow or yellow berries with numerous tiny (0.5 mm) seeds, embedded in is necessary for successful establishment). Testing this would a fl eshy sticky pulp ( Bouman and Meijer, 1994 ; S. Bellot, personal require phylogenetic information at the genus level for para- observation). Birds have been observed eating the fruits of Pilo- sites and other angiosperms. styles ingae in Brazil ( Brasil, 2010 ). Apodanthaceae fl owers are sessile, minute, and dark-red or Animal pollination is overrepresented in parasitic angio- yellow (depending on the species), with nectar offered in minute sperms — As shown in Table 1 , parasitic angiosperms are animal- quantities (this study) on a shallow nectary cushion at the base pollinated, while among the remaining angiosperms at least of the fl owers. Pollination by short-tongued nectar-foragers was 24 000 species are wind-pollinated, with wind pollination hav- therefore expected, and as demonstrated here, at least Pilostyles ing evolved at least 65 times ( Linder, 1998 ). Ecological reasons aethiopica is indeed pollinated by short-tongued fl ies. An earlier for the absence of wind pollination among parasites may be study of this species also reported fl ies as fl ower visitors, namely their occurrence at ground level (root parasites), on the stems of Drosophila, Sciaridae, Psychodidae, and Cecidomyiidae (Blarer trees (e.g., Apodanthaceae), or within leafy tree crowns (e.g., et al., 2004 ), but provided neither details on their foraging behav- mistletoes), where pollen export and import by wind might be ior on the fl owers nor whether they visited both sexes. relatively diffi cult. All animal pollinators are represented among The fl ower scent likely consists of ethanol and related com- parasites, ranging from ants, via birds, to bees, butterfl ies, bee- pounds in addition to fruity compounds as known from other fl y- tles, and fl ies, to shrews ( Figs. 3, 4 ; Table 1 and its references). pollinated fl owers, including Araceae (Kite and Hetterscheid, Among seed dispersal modes, dispersal by animals predomi- 1997), Schisandraceae (Yuan et al., 2008), Trollius (Ibanez et al., nates, although not as pervasively as animal pollination. 2010 ), but also wasp-pollinated fl owers ( Johnson et al., 2009 : Drypetes natalensis ). Females of Calliphoridae (blow-fl ies), that Conclusions — In conclusion, the absence of wind pollina- also visit carrion, are attracted by such scents (e.g., Jürgens et al., tion is clearly a feature uniting parasitic angiosperms (not noted 2006). No insect eggs or larvae have been reported from Apodan- by Molau, 1995). Dioecy appears to be more common among thaceae fl owers, nor were any found in our study, and the fl ies parasites than it is among other fl owering plants, but phyloge- therefore must come for pollen or nectar. In an Australian species netic resolution so far is insuffi cient to infer shifts to and from of Pilostyles , a Brazilian species of Pilostyles , and the Panama- dioecy. The African and Iranian species of Apodanthaceae stud- nian Apodanthes caseariae , wasps have been observed on the ied here are dioecious and pollinated by fl ies. Resolving whether fl owers (Dell and Burbidge, 1981; Brasil, 2010), with the Pana- a combination of animal pollination, perennial life history, and manian wasp being identifi ed as Pepsis menechma Lepeletier, dioecy facilitated the evolution of parasitism or evolved subse- 1845 (C. Galdames, Smithsonian Tropical Research Institute, quent to the evolution of parasitism will require ancestral trait Panama; personal communication). reconstructions in several of the 13 lineages. At the moment, we are far from understanding the ecological and evolutionary in- Is dioecy overrepresented in parasitic angiosperms? — Di- teractions among hosts, parasites, their pollinators and dispers- oecy has evolved in fi ve of the 13 parasitic angiosperm clades, ers even in the best-studied systems, Cuscuta , Viscum , or the amounting to 456 species or 10% of 4510 parasitic species ( Table 1 ; economically important pests in Orobanchaceae, and compared Appendix S1). Among nonparasitic angiosperms, 14 164, or 6%, to earlier reviews (Kuijt, 1969; Molau, 1995), ecological data are dioecious, assuming a total of 240 000 angiosperm species have been slower to accumulate than molecular ones. ( Renner and Ricklefs, 1995 ). These estimates have unknown er- ror margins. 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