Flora 215 (2015) 75–83 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.com/locate/flora The status of the mistletoe genus Dufrenoya Chatin (Amphorogynaceae) with a specific focus on Nepal Mohan P. Devkota a , Jill Macklin b,1 , Daniel L. Nickrent c,∗ a b c Department of Botany, Amrit Campus, Tribhuvan University, Kathmandu, Nepal Department of Botany, Trinity College, The University of Dublin, Dublin 2, Ireland Department of Plant Biology, Southern Illinois University, Carbondale, IL 62966-6509, USA a r t i c l e i n f o Article history: Received 21 February 2015 Received in revised form 29 April 2015 Accepted 22 July 2015 Edited by Bohumil Mandák Available online 31 July 2015 Keywords: Parasitic plant Santalales Santalaceae Phylogeny Taxonomy Himalaya a b s t r a c t The genus Dufrenoya Chatin (Amphorogynaceae) is a rarely collected and studied aerial parasite found of southeastern Asia. Our goal is to provide a modern treatment of the genus for Nepal where two species occur: D. platyphylla (the type of the genus), and D. granulata. Fresh, fixed, and dried material of the former species and herbarium material of the latter was examined and used to prepare morphological descriptions. In addition, the ITS rDNA region was sequenced and phylogenetic analyses conducted on five genera of Amphorogynaceae. Strong support for the sister relationship between Dufrenoya and Phacellaria was obtained. The complex nomenclatural history of Dufrenoya is reviewed and discussed in the context of intergeneric relationships in the family. The morphology and anatomy of flowers and fruits are photographically illustrated and discussed. Collections of both species plotted on a map of Nepal showing ecological zones and physiographic regions indicates Dufrenoya is primarily distributed in the Middle Mountains region. © 2015 Elsevier GmbH. All rights reserved. 1. Introduction The most familiar mistletoe to Europeans is Viscum album (Zuber, 2004), but the mistletoe habit has actually evolved independently in five Santalales families: Misodendraceae, Loranthaceae, Santalaceae, Viscaceae, and Amphorogynaceae (Nickrent et al., 2010). The latter three families are all considered Santalaceae s. lat. by APG III (2009); however, here they will here be considered as distinct following Nickrent et al. (2010). Amphorogynaceae is sister to the economically important mistletoe family Viscaceae and is remarkable in that it contains plants with a wide range of trophic modes and habits. For example, the genera Choretrum and Leptomeria are root hemiparasites whereas some Dufrenoya and all Phacellaria are best referred to as mistletoes. Furthermore, Dendromyza is a stem parasitic liana (dendroparasite, Vidal-Russell and Nickrent (2008) see Fig. 4D) that attaches first to the host with a primary haustorium and then forms two kinds of shoots: leafy non-twining as well as squamate, twining, haustorial stems. Some Dendrotrophe species can be terrestrial shrubs or woody climbers ∗ Corresponding author. E-mail addresses: himalayanforum@gmail.com (M.P. Devkota), macklinjill@gmail.com (J. Macklin), nickrent@plant.siu.edu (D.L. Nickrent). 1 Current address: 113 Woodbine Park, Raheny Dublin 5, Ireland. http://dx.doi.org/10.1016/j.flora.2015.07.009 0367-2530/© 2015 Elsevier GmbH. All rights reserved. that parasitize the roots of host plants whereas others are dendroparasitic climbers. But unlike Dendromyza, Dendrotrophe does not form stems with secondary haustoria. Species such as Dendrotrophe buxifolia and D. varians that can exist as either stem or root parasites are called amphiphagous (Der and Nickrent, 2008). The amphiphagous condition has also been reported for Daenikera (Stauffer, 1969) where the host is Amphorogyne, both endemic to New Caledonia. Because these are sister genera, this may represent a case of adelphoparasitism (parasite evolved from its host), rarely documented in flowering plant parasites. The stem morphologies seen across all 11 species of Dufrenoya are actually quite varied such that some are mistletoes of the “Viscum album” type according to Stauffer (1969). In Dufrenoya the stems may be erect or pendulous and in species such as D. papillosa, D. poilanei, and D. sessilis, at least some twining stems can be seen. But unlike Dendromyza, these twining stems do not form secondary haustoria. This is contrary to the description in Kuijt (2015) as well as Danser (1940) who said (p. 142): “Hylomyza [Dufrenoya, see below] agrees, in its mode of growing, with the non-climbing Cladomyza species and also with Phacellaria.” The genus Dufrenoya was first proposed by Chatin (1860), but in fact the plant upon which this name is based had been discovered and described 35 years earlier. Plants now assigned to Dufrenoya platyphylla have previously been included in five 76 M.P. Devkota et al. / Flora 215 (2015) 75–83 different genera, and the complex and turbulent nomenclatural history is briefly summarized here. Francis Buchanan-Hamilton first collected Dufrenoya in 1802 in Nepal under the name Viscum latifolium Buch.-Ham ex Don (1825). This mistletoe was named V. platyphyllum by Sprengel in 1827 and V. heteranthum Wallich ex A.P. De Candolle in 1830. The Wallich collection (no. 488) from the Kathmandu district is the type for the species and genus. Not accepting placement in the genus Viscum, Joseph D. Hooker named this mistletoe Henslowia heterantha Hook. f. ex A.P. De Candolle (1857), within a genus of Santalaceae named in 1951 by Blume. Based on the Wallich specimen, Gaspard A. Chatin erected the genus Dufrenoya in 1860 (commemorating his former teacher Pierre-A. Dufrénoy), making the new combination Dufrenoya heterantha Chatin. Based on nomenclatural rules and differences in fruit structure, Benedictus Danser (1940) replaced Henslowia with Dendrotrophe and created three new genera to accommodate the remaining species: Cladomyza, Dendromyza, and Hylomyza. Citing all the previous names as synonyms, Danser named Hylomyza platyphylla (Sprengel) Danser based on Viscum platyphyllum. The combination Dendrotrophe heterantha Henry and Roy (1969) apparently went unnoticed by those who prepared the posthumous manuscript by Stauffer (1969). Here the genera segregated from Hylomyza by Danser were retained, but Dufrenoya was reinstated, thus making Hylomyza a synonym. The earliest name, Dufrenoya latifolia, could not be used because V. latifolium Buch.Ham. was a later homonym for an illegitimate name (V. latifolium Swartz) and a synonym of Phoradendron piperoides (V. latifolium Lam.). The name D. heterantha Chatin could not be used because it is based on V. heteranthum Wall. which is a nomen nudum. Thus the correct new combination published was Dendrotrophe platyphylla (Sprengel) Stauffer. Finally, the most recent (unfortunate) nomenclatural activity on this taxon was the publication in the Flora of China of D. platyphylla (Sprengel) N.H. Xia et M.G. Gilbert. Nepal is a global biodiversity hotspot that contains over 7000 species of vascular plants and efforts to document its flora are ongoing (Bista et al., 2001; Hara et al., 1978, 1982; Hara and Williams, 1979; Press et al., 2000). At present, Santalales have yet to be treated. In general, Dufrenoya is poorly studied and relatively few collections of this genus exist within and outside of Nepal. Moreover, prior to the photos taken for this project, no photographs of living Dufrenoya plants were available. The purpose of this paper is to assemble available information and report new observations on two species of Dufrenoya from Nepal, D. platyphylla and D. granulata, with the goal of clarifying their taxonomic status. 2. Materials and methods rigidula (KP828446). In addition to the above species, complete ITS sequences for three ingroup taxa were obtained from NCBI Genbank: Dendromyza reinwardtiana (DQ333870), D. varians (DQ333871), and Leptomeria cunninghamii (GU256865). The outgroups used were Exocarpos cupressiformis (HM116973), E. sparteus (GU256864) and Santalum album (JX856495). A manual alignment (available upon request) was made using SeAl (Rambaut, 2007). Maximum parsimony (branch and bound search) and bootstrap analyses were conducted with PAUP* (Swofford, 2002). 2.2. Field work The site where Dufrenoya platyphylla was observed and collected is located near Kande village in the Western Region of Nepal (Table 1). One of us (MPD) made ten visits between February and September in 2010 as well as January and June 2012. Photographs, herbarium specimens, and alcohol fixed samples were obtained of flowering and fruiting plants. The host was a 12 m tall Castanopsis indica tree located at the margin of an agriculture terrace. Multiple mistletoe infections that measured from <50 cm to 1.5 m in diameter were observed on the host tree. 2.3. Herbarium work Past collections of Dufrenoya in Nepal were examined and in some cases photographed by MPD. Two species are currently known from the country: D. platyphylla and D. granulata. Herbarium material was studied for both species whereas fresh material was available only for the former. The visited herbaria were Royal Botanic Garden Edinburgh (E), Natural History Museum in London (BM) and Royal Botanic Gardens Kew (K) from October to December, 2011, the National Herbarium in Kathmandu (KATH) in 2012 and 2015, and University of Tokyo Herbaria (TI) in September 2013. In addition to physical visits, virtual visits to the following online herbaria were made: B, BM, GH, Kew, P, L, E, and NY. Photos and metadata associated with these collections were assembled. 2.4. Anatomical work Alcohol fixed material was used for dissection and photography using a stereomicroscope. Flowers and fruits were also prepared using standard paraffin embedding techniques, sectioned with a rotary microtome, and stained with saffranin and fast green. The slide were observed and photographed using an Olympus CH30 compound microscope fitted with an Olympus DP-20 digital camera. 2.1. Molecular work 2.5. Distribution mapping Genomic DNA was obtained from herbarium specimens using a 2X CTAB protocol (Doyle and Doyle, 1987) followed by a clean-up step using spin columns (Concert Rapid PCR Purification System, Life Technologies). A portion of the 5.8S rDNA and ITS-2 was PCR amplified using the ITS3 forward primer (White et al., 1990) and the AB102 reverse primer (Sun et al., 1994). Amplification utilized a standard PCR protocol with 5 M betaine added (20% of final volume). Thermal cycle parameters were 1 min 97 ◦ C, 2 min 50 ◦ C, and 3 min 72 ◦ C for 40 cycles. Cycle sequencing followed the manufacturer’s protocol (PerkinElmer Cetus) and the reactions were run on an ABI 377 automated DNA sequencer at the Royal Botanic Gardens, Kew. The six newly sequenced taxa and their Genbank accession numbers are: Dendromyza ledermannii (KP828443), D. buxifolia (KP828444), Dendrotrophe varians (KP828445), Dufrenoya robusta (KP828448), Dufrenoya sessilis (KP828447), and Phacellaria Attempts to obtain coordinates for all Dufrenoya collections made in Nepal was made difficult because some herbarium specimen labels did not provide sufficient information to accurately locate the collection site. Moreover, some labels only indicated the overall excursion track taken by the botanical team. The Flora of Nepal website (http://padme.rbge.org.uk/floraofnepal/) was utilized to check place names, locations, and collectors. Latitude/longitude positions were in some cases inferred from the place name and elevation. Following this procedure, 12 discrete records of D. platyphylla and 8 records of D. granulata (Table 1) were plotted using Google My Maps. A map showing ecological zones and physiographic features for Nepal (Lillesø et al., 2005) was used as a layer in Adobe Photoshop and the dots from the Google map overlayed upon it. Lat/long 1770 m 2500 m 900–1000 m 1275 m 1338 m 2307 m 2100 m 2000 m 1676 m 1707 m 1708 m 1970 m 2080 m 1920 m 2121 m 2000 m 2000 m 2300 m 2300 m 1920 m 27 47 17.01N, 85 20 29.73E 27 42 01.14N, 86 01 06.25E 27 46 43.40N, 86 11 40.21E 27 16 03.01N, 85 56 59.02E 27 33 05.13N, 85 07 35.16E 27 36 29.34N, 85 05 39.65E 27 27 06.07N, 87 13 19.81E 27 28 22.21N, 87 37 55.04E 27 24 00.00N, 87 54 00.00E 28 19 56.48N, 83 48 34.67E 28 17 43.96N, 83 49 21.00E 28 16 56.51N, 84 14 11.53E 27 34 06.55N, 86 21 14.76E 27 26 50.82N, 87 12 2.93E 27 32 37.28N, 87 36 51.89E 27 15 01.22N, 87 56 07.97E 27 24 47.70N, 87 53 21.75E 27 35 11.37N, 87 47 40.15E 27 32 57.80N, 87 46 04.94E 28 18 10.40N, 83 48 52.90E Pararil–Tokha Chandeswori On way–Kalinchowk Tamba Koshi–Malekhu–Kosikhet–Pikhuti Dhungre Bhanjayng, Sindhuli Bhimphedi Trisuwan Rajpath, Daman Bhotebas to Chichila Papung–Dongen–Mewa Khola bridge–Sawadin Yamphudin–Helok Between Landruk [Ghandruk] and Dhampus, NW of Pokhara Kande village near Lumle Pasgam Patkare–Bhandar Bhotebas–Gogane–Chichila Dongen Birwa (near Chyangthapu) Ghatte–Khebang Ila Danda–Solap Thakpa Bazaar–Suketar (Tamur bridge)–Ramsyang Pati Lumle, above Agricultural Centre Locality District Zone Bagmati Janakpur Janakpur Janakpur Narayani Narayani Koshi Mechi Mechi Gandaki Gandaki Gandaki Janakpur Koshi Mechi Mechi Mechi Mechi Mechi Gandaki Region Central Central Central Central Central Central Eastern Eastern Eastern Western Western Western Central Eastern Eastern Eastern Eastern Eastern Eastern Western Date 488 381 8340419 2040 s.n. 5298 93 9263043 982 2387 475 5927 8530777 8840091 25710 6301228 6301227 6301226 9263134 492 Wallich N. Malla S.B. & Rajbhandari S.B. Ohba et al. Manandhar N.P. & Adhikari M.K. Buchanan Stainton J.D.A. Long D.G. et al. Suzuki M. et al. Williams L.H.J. Sayers C.D. Devkota M. Stainton Sykes & Williams Ohba et al. Suzuki M. et al. Beer L.W. Hara H., Kanai H., Kurosawa S. Hara H., Kanai H., Kurosawa S. Kanai H. et al. Suzuki M. et al. Dawson G. D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. platyphylla D. granulata D. granulata D. granulata D. granulata D. granulata D. granulata D. granulata D. granulata 1830 11 October 1960 27 August 1983 3 June 1979 1802 20 March 1966 20 September 1991 14 May 1992 27 June 1969 10 April 1975 30 June 2001 25 June 1954 5 August 1985 8 July 1988 5 November 1975 27 November 1963 28 November 1963 9 November 1963 23 May 1992 25 December 1975 Number Collector Dufrenoya species Table 1 Collection information for the Dufrenoya species from Nepal examined in this study Kathmandu Dolakha Dolakha Ramechap Makwanpur Makwanpur Sankhuwasabha Taplejung Taplejung Kaski Kaski Lamjung Ramechap Sankhuwasabha Ilam Panchthar Panchthar Taplejung Taplejung Kaski Elevation M.P. Devkota et al. / Flora 215 (2015) 75–83 77 3. Results and discussion 3.1. Molecular phylogenetic results The molecular phylogenetic investigation of (Der and Nickrent, 2008) sampled all genera of Amphorogynaceae and all intergeneric relationships were fully resolved. Dufrenoya is strongly supported was sister to Phacellaria using 18S rDNA, rbcL and matK and that clade was sister to another composed of Dendromyza and Dendrotrophe. In contrast to these relatively slowly evolving genes, the ITS region (internal transcribed spacers 1 and 2 plus 5.8S rDNA) is rapidly evolving, particularly in mistletoes (Ashworth, 2000; Molvray et al., 1999; Nickrent et al., 2004). Of the total 819 characters in this alignment of Amphorogynaceae, 252 (30%) are parsimony informative. In general, both ITS-1 and ITS-2 are significantly more variable than the 5.8S rDNA region and ITS-1 is more variable than ITS-2. Although, Phacellaria clearly shows sequence similarity to Dufrenoya, its rate of evolution is higher (more substitutions and insertion/deletion mutations). Such elevated rates are also seen in the dwarf mistletoes (Arceuthobium, Viscaceae), which like Phacellaria are also squamate indicating a convergent morphological and molecular evolutionary pattern. Branch and bound analyses found one tree of length 810 (Fig. 1). Amphorogynaceae was strongly supported as monophyletic (99% BS). A Dendrotrophe/Dendromyza clade (67% BS support) is sister to another clade composed of Dufrenoya, and Phacellaria (100% BS support). These two clades were sister to Leptomeria (from Australia). That Dufrenoya and Phacellaria are closely related is also reflected in fruit and seed characters (Danser, 1940). Thus, the decision by Xia and Gilbert (2003) to lump Dufrenoya into Dendrotrophe was ill-advised, especially since they recognized the genus Phacellaria as distinct. Dendrotrophe and Dendromyza were not monophyletic. The Genbank sequence DQ333871 (voucher Calvin and Wilson B0215) appears most closely related to D. buxifolia, thus the specimen could be misidentified. Clearly, more work is needed to determine whether Dendrotrophe and Dendromyza are distinct genera, a sentiment also expressed by Kuijt (2015) who provisionally lumped the former into the latter. 3.2. The genus Dufrenoya In the posthumous publication by Stauffer (1969), no generic description was given, only citations of the original paper by Chatin (1860) and Danser (1940) who described the genus as Hylomyza. Dufrenoya can be distinguished from Dendrotrophe by the presence of umbellate vs. racemose male inflorescences. It differs from Dendromyza in fruit features such as an endocarp with apical chambers vs. a unilocular endocarp or with chambers at the base. Macklin (2000) provided detailed generic and specific descriptions for the 11 recognized species of Dufrenoya. Descriptions of the four species native to Thailand were given in Macklin and Parnell (2002). This information and novel observations were utilized in the following generic description. 3.2.1. Dufrenoya chatin 3.2.1.1 Type species: Df. platyphylla (Sprengel) Stauffer Glabrous, aerially parasitic mistletoes with or without twining stems; stems spreading, erect or pendulous, if twining climbing but not forming secondary haustoria. Stem surface smooth or pustulate, juvenile and older stems distinctly different, bracteate at base. Bark on older growth with longitudinal fissures. Leaves alternate, simple, without stipules, petiolate, elliptic, obovate, or spatulate, coriaceous, venation basal actinodromous, with 3–11 primary veins. The genus is dioecious or in some species possibly gynodioecious. Male inflorescence an umbel of 5–7 flowers at the apex of a bracteate peduncle; peduncles of varying lengths, single or 78 M.P. Devkota et al. / Flora 215 (2015) 75–83 Fig. 1. Single tree (length 810) obtained from branch and bound analysis of ITS sequences from five genera of Amphorogynaceae. Maximum parsimony bootstrap consensus (1000 replications) values shown above nodes. Consistency index minus uninformative sites 0.6964, retention index 0.6531, rescaled consistency index 0.5177. clustered in groups, arising from 1 to 2 year old stems, sometimes from brachyblasts; peduncles with tightly grouped bracts at the base and apex, apical bracts forming an involucre, also sometimes bearing bracteate lateral flowers. Male flowers in lateral position in axils of bracts, central flower ebracteate; calyx lobes absent, corolla lobes (petals) valvate, cuculate at apex, glandular hairs sometimes present on interior surface; stamens equal in number to petals, antipetalous, anthers dithecal, each theca with two locules (bilocular, hence tetrasporangiate), locules isomerous or unequal with the posterior larger than the anterior, dehiscence transverse or longitudinal; nectary flat to concave, fleshy, smooth and scarcely 5-lobed; style rudiment variously developed. Female inflorescence pedunculate, 1-flowered. Female flower with 5 deltoid, valvate petals, staminodes present or not, disk flat, 5-lobed, ovary inferior, style short, cylindrical, style with 5 papilliform spreading stigmas, placenta convex, 5–6-lobed, placental column long, not twisted. Fruit an ellipsoid, ovoid or obovoid drupe borne singly at apex of peduncle; petals persistent at apex, erect or not; exocarp thin, mesocarp consisting of short or long membranous strands that wrap around the endocarp or do not; endocarp forming a 1-seeded pyrene, hard or not, if the latter then splitting easily, externally longitudinally grooved, internally 5–7 chambered at apex and base, incompletely so in central part; seed with same number of lobes as endocarp chambers. Dufrenoya contains ca. 11 species distributed in northern India, Nepal, Bhutan, southern China, Myanmar, Thailand, Laos, Vietnam, Indonesia (Java, Sumatra) and Malaysia (peninsular and western Borneo). 3.3. Dufrenoya species in Nepal Neither Danser (1940) nor Stauffer (1969) provided descriptions for the two species of Dufrenoya that occur in Nepal, only citing the original protologues. The first of the two species is D. platyphylla (Sprengel) Stauffer which also occurs in India, Bhutan, Myanmar, and Thailand (the latter a new record reported in Macklin, 2000). The second species, D. granulata (Hook. f.) Stauffer, is also found in India, Bhutan, and southern China. These mistletoes occur in submontane and montane regions, particularly in evergreen oak and oak-pine forests between 1500 and 2500 m elevation. The most frequent hosts are in Fagaceae (Quercus, Castanopsis, and Lithocarpus) and less frequently on other genera (e.g., Rhododendron, Schima, and Symplocos). 3.3.1. Dufrenoya platyphylla (Sprengel) Stauffer Type: Wallich 488 (Holotype of Viscum heteranthum K!; Isotypes E!, L!, P!, TCD!) Nepal. Stauffer, Vierteljahrsschrift der Naturforschenden Gesellschaft in Zürich 114: 70 (1969); Grierson et Long, Flora of Bhutan 1.1: 143 (1983); V. platyphyllum Sprengel, Linnaeus Systema Vegetabilium XIV (1824), Curae Posteriores: 47 (1827); DC Prodromus 4: 279 (1830); V. latifolium Buchanan-Hamilton ex D. Don, Prodromus Flora Nepalensis: 142 (1825); V. heteranthum Wall ex DC, Prodromus 4: 279 (1830); H. heterantha Hook. f. ex A.DC, Prodromus 14: 632 (1857); Kanjilal, Flora of Assam 4: 130 (1940); D. heterantha Chatin, Comptes rendus Acadamie Paris 51: 657 (1860); H. platyphylla Danser, Nova Guinea New Series 4: 145 (1940); D. heterantha Henry et Roy, Bulletin of the Botanical Survey of India 10: 274 (1969) syn. nov. D. platyphylla (Sprengel) N.H. Xia et M.G. Gilbert, comb. nov. Flora of China 5: 216 (2003). A large shrub (mistletoe), up to 2 m in diameter; stems erect or pendulous, not twining, branched, often 3–4 from a single node, spreading on the trunks and branches of the host tree and attaching by means of a large network of epicortical roots. Juvenile stems terete, pustulate (verruculose), flattened towards the apex, slightly keeled, bracteate at the base at junction with older stem; older stems distinctly different, pale in color, with longitudinal fissures. Leaves coriaceous, (30−) 51(−97) × (15−) 31(−74) mm, elliptic, ovate, obovate, apex obtuse to sub-acute, base obtuse, attenuate or cuneate, margin entire, upper surface shiney, smooth, lower surfaces dull, primary veins 5–7, raised on the upper surface, secondary veins fine, arising at approximately right angles, opposite secondaries, percurrent; young leaves shiny dark brown; petiole angular, 8.0–10.0 × 1.5 mm. Male inflorescences in leaf axils and along internodes; peduncles up to 8 in a cluster, on juvenile stems and on short shoots arising from the older stems, terete, 2.0–6.0 × 0.5–0.7 mm, with a series of small bracts at the base, larger ones (ca. 1.0 mm) spiralling M.P. Devkota et al. / Flora 215 (2015) 75–83 79 Fig. 2. Flowering and fruiting shoots of Dufrenoya platyphylla, all from Kande village, Kaski district, Ganddaki zone, Nepal (Devkota s.n.). A — shoot with unopened flower buds (January 2012); B — shoots with male flowers in full anthesis (April 2012); C — closer view of A showing the cluster of male flowers at the peduncular apex; D — shoot with male flower in full anthesis (April 2012); E — fruiting shoot. upward to ca. half way up the peduncle, at its apex bearing a single involucre composed of ca. 6 quincuncially arranged, broadly ovate, ciliolate bracts, 0.8–1.0 × 0.8–0.9 mm, each bract bearing a flower at its base, apex terminated by a central flower. Male flowers greenish, subsessile, with receptacle gradually attenuate, becoming extremely narrow at base, lateral flower arising from pockets at the base of their subtending bracts; central flower ebracteate, globose, radially symmetrical, lateral flowers asymmetrical (by compression), turbinate; petals 5–6, deltoid, 1.4–1.5 × 1.0 mm, interior surface densely lined with hairs; filaments short, 0.2–0.3 × 0.1–0.2 mm, slightly dilated at the base, anthers basifixed, 0.2–0.3 × 0.4–0.5 mm, dithecal, lobes unequal in size, anterior shorter and broader than the posterior, dehiscence transverse; post-staminal hairs emanating from glandular cells on petal at base of filament, connecting (postgenitally) to anther; nectary disk 5–6 lobed, flat, apiculate in the center (a pistillode?). Female inflorescences sessile or shortly pedunculate. Female flowers green, buds distinctly turbinate, petals 5–6, elongate-ovate, apex acuminate, 1.0 × 0.6 mm, receptacle broad, 1.4 × 1.0 mm, staminodes present or absent, filaments 0.2 × 0.2 mm, anthers 0.3 × 0.4 mm, style short, broad, 0.3 × 0.3 mm, stigma 3–5 lobed, lobes fleshy, weakly papilliform. Fruit 3.8–5.0 × 3.0–4.0 mm, globose or slightly obovoid, persistent petals forming an apical mound, changing from green to pale yellow, to pink or purple upon ripening, exocarp smooth, thin, mesocarp composed of short, membranous, randomly oriented strands, endocarp hard, not splitting easily, globose or slightly obovoid, 2.1–2.5 × 1.6–1.8 mm, beaked at apex, apiculate at the base, distinctly 4–6 lobed, lobes rounded at the apex, internally 4–6 chambered at the apex and the base, incompletely so in the middle, bearing a crown of fibrovascular fibers at the apex. 3.3.2. Dufrenoya granulata (Hook. f. et Thompson) Stauffer Type: Hook. f. (Holotype of Henslowia granulata K!; Isotypes E!, K!, L!, NY!, TCD!; P!) India (Sikkim, Khasia). Stauffer, Vierteljahrsschrift der Naturforschenden Gesellschaft in Zürich: 70 (1969); Grierson et Long, Flora of Bhutan 1.1: 142 (1983); H. granulata Hook. f. ex A.DC., Prodromus 14: 632 (1857); Hook. f., Flora of British India 5: 232 (1886); Kanjilal, Flora of Assam 4: 129 (1940); [H. granulata sensu Collett et Hemsley, Journal of the Linnean Society 28: 121 (1890)]; Hylomyza granulata Danser, Nova Guinea New Series 4: 145 (1940); Dendrotrophe granulata Tam, Flora Reipublicae Popularis Sinicae: 71 (1988) syn. nov. Erect and pendant shrub (mistletoe), stems not twining. Juvenile stems angular, flattened, densely pustulate, bracteate at the base at junction with older stem; older branches terete, bark fissured. Leaves coriaceous, (14−) 27(−36) × (14−) 18(−20.0) mm, elongate-obovate, apex rounded, tip sometimes mucronate, base attenuate, upper surfaces weakly convex, smooth, shining, lower surface rough, dull, margins thin, undulating, completely smooth and flattened, primary veins 5–9, raised on the upper surface, secondary veins indistinct; petiole long, 2.0–3.5 × 1.5 rom, upper surface flattened, lower surface angular. Male inflorescences on juvenile shoots and those of second years growth, usually 2–3 in a cluster, 6-flowered, very shortly pedunculate, peduncles 0.5–0.6 × 1.0–1.2 mm, and involucre of 2–3 bracts at the base and a single involucre of 5, quincuncially arranged bracts at the apex, bracts large, broadly ovate, 1.0–1.5 × 1.0–2.0 mm, papyraceus, clearly divided into two areas, inner region thick, hard, margin thin and entire or sparsely ciliate. Male flower buds turbinate, green, tinged red, pedicel decurrent with receptacle, 0.8–1.5 × 0.8 mm, petals 5, broadly ovate, 1.0–1.2 × 1.4–1.6 mm, fleshy, apiculate on the inner surface at the apex, glandular hairs present, filaments long, narrow, 0.3 × 0.2 mm, anthers dorsifixed, large, broad, 0.3–0.4 × 0.6 mm, bithecate, lobes isomerous, dehiscence longitudinal, nectary disk flat, distinctly 5-lobed, lobes broad, pointed, style rudiment absent. Female flower unknown (description taken from fruiting specimen), tepals elongate — deltoid, 1.0–1.1 × 0.7–0.8 mm, apiculate on the inner surface at the apex, staminodes absent, stigma occupying the entire center of the flower, sessile, large, entire, weakly papillose, slightly 80 M.P. Devkota et al. / Flora 215 (2015) 75–83 Fig. 3. Morphology and anatomy of male flowers from Dufrenoya platyphylla, all from Kande village, Kaski district, Ganddaki zone, Nepal (Devkota s.n.). All from alcohol fixed material. A — inflorescence showing cluster of pedunculate male umbels; B — top view of umbel showing globose central flower surrounded by compressed lateral flowers; C — open male flower showing six petals and dehiscing anthers; D — L.S. male flower bud showing interlocking valvate petal margins, stamen and central disk; E — closer view of valvate aestivation of petal margins, postgenitally coherent by their interlocking petal epidermal cells; F — hairs lining the inner surface of the petals; G — stamens removed from flower showing the dithecal anthers, each with two unequal thecae (posterior larger than the anterior); H — L.S. flower showing the post-staminal hairs, their basal portion of swollen, glandular cells as part of the perianth (cf. Santalum album, Fig. 18 in Rao, 1942), and the flat central disk; I — narrow pedicel where it enters the pit at the base of the involucral bract. depressed in the center, nectary flat or convex, angular. Fruit 4.5–5.0 × 4.5–5.0 mm, globose, persistent petals erect, red to dark purple upon ripening, exocarp smooth, coriaceous, mesocarp composed of short, membranous, randomly oriented strands, endocarp splitting easily, obovoid, 2.7–3.0 × 1.8–2.0 mm, apiculate at the base and the apex, distinctly 5-lobed, internally 5-chambered at the apex and the base, incompletely so in the middle, not bearing a crown of fibrovascular bundles. The following features can distinguish the two species of Dufrenoya found in Nepal. The leaves of D. platyphylla are significantly larger than D. granulata (average length 51 vs. 27 mm). Both species have variation in leaf shape (elliptic to obovate) but D. platyphylla also shows ovate leaves. The number of male inflorescence pedicels and their lengths are different between the two species (longer and more numerous in D. platyphylla). Male flowers are subsessile in D. platyphylla and pedicellate in D. granulata. Anther lobes are unequal with transverse dehiscence in D. platyphylla whereas in D. granulata they are isomerous with longitudinal dehiscence. The involucral bracts at fruiting time are larger in D. granulata and may be slightly accrescent. Finally, the endocarp of D. platyphylla has an apical crown of fibrovascular fibers whereas D. granulata lacks these. Additional floral and fruit features appear to show differences; however, more work is required to confirm them. 3.4. Dufrenoya morphology and anatomy The arrangement of male flowers while still in bud is shown in fresh condition (Fig. 2A and C) and in fixed material (Fig. 3A and B). The globose central flower is surrounded by laterals that are distinctly flattened dorsally and compressed laterally against each other and the central flower. The term “fungiform” has been used to describe their shape, although here turbinate (top-shaped) was used. As described above, a male flower in D. platyphylla arises from a pocket formed at the base of its subtending bract, a feature apparently not previously described. The floral pedicel becomes extremely narrow at this point of entry (Fig. 3I). Because only one fruit is ever observed within one involucre (Fig. 4A), it is possible that these lateral flowers only serve as males and abscise soon after anthesis and pollen dehiscence. The valvate petals are interlocked at their margins by hairs (Fig. 3D and E). Open flowers (Figs. 2B and D, 3C) are minute, thus the aspect of the shoot in full anthesis differs little from when it is in bud stage. Given the flower size, color, presence of a glandular disk, and lack of a discernable smell, small insects are the likely pollinators. Indeed, small flies (at present unidentified) were seen visiting flowers at the Kande Village site by one of us (MPD). The hairs often seen associated with stamens in Amphorogynaceae and related families are quite recognizable but have received various names. They have been called staminal hairs (Ewart, 1892), glandular hairs (Macklin and Parnell, 2002), and post-staminal hairs (Kuijt, 2015) and apparently exude a viscous, oily fluid. The post-staminal hairs seen in D. platyphylla (Fig. 3H) look remarkably similar those from S. album illustrated by Rao (1942) his Fig. 18, complete with a swollen basal portion containing a large nucleus. It should be noted that in D. platyphylla, the interior surface of the petals is lined with M.P. Devkota et al. / Flora 215 (2015) 75–83 81 Fig. 4. Morphology and anatomy of fruits from Dufrenoya platyphylla, all from Kande village, Kaski district, Ganddaki zone, Nepal (Devkota s.n.). All from alcohol fixed material. A — fruits borne on peduncles, one fallen revealing subtending involucre; B — L.S. of fruit apex showing persistent petals, staminodes, and whithered papilliform stigma; C — L.S. of fruit apex showing glandular disk and the membranous mesocarp; D — fruit X.S. showing thin exocarp, fleshy mesocarp, 6-lobed endocarp enclosing the seed; E — endocarp dissected from fruit showing the longitudinal grooves; F — endocarp dissected from fruit, slightly stained with methylene blue, showing the apical beak and the crown of fibers attached to endocarp; G — fruit L.S. stained with methylene blue showing fiber structure; H — tangential section of fruit, stained with methylene blue, showing clusters of brachysclereids just below exocarp and fibers in the mesocarp below the sclerenchyma clusters; I — endocarp composed of brachysclereids (somewhat disrupted by microtomy). hairs (glandular?) that are distinct from the post-staminal hairs (Fig. 3F). The anther morphology in Dufrenoya varies between species. For D. platyphylla and six other species, the anterior lobes are smaller than the posterior and dehiscence is transverse (Fig. 3G). In three species (D. oresitropha, D. poilanei, and D. robusta), the lobes are equal in size and shape (isomerous) and dehiscence is longitudinal. Anther lobe type and dehiscence roughly correlates with pollen exine morphology where isomerous lobes that dehisce longitudinally have echinate pollen exines whereas unequal lobes with transverse dehiscence have rugulate pollen exines (Macklin, 2000). This correlation does not hold up for D. granulata and D. papillosa which have the inverse relationship. Dufrenoya is usually described as being dioecious, but there is evidence that its sexual system is more complex. In Fig. 28 of Danser (1955) the flower of D. oresitropha (as Hylomyza) is labeled as “hermaphrodite (or pseudo-hermaphrodite)”. Moreover, all descriptions of D. granulata indicate that female flowers are unknown; however, given that the isotype at K collected by Hooker and Thompson from Sikkim India bears fruits, these flowers could be bisexual. When observing fruits of D. platyphylla, some clearly have staminodes (Fig. 4B) whereas others apparently lack them (Fig. 4C), and these two fruit types came from the same individual. So in this case, the structures could be functional stamens or staminodes. If one individual bears fully functional bisexual flowers as well as female flowers and in the same population completely male individuals exist, this condition would be polygamodioecy. If the structures in some of the female flowers are staminodes, the sexual condition is dioecy but with those females being transitional between bisexual and unisexual. The fruits in Dufrenoya and other Amphorogynaceae are the most complex in the sandalwood order, and their functional morphology is still incompletely understood. Substantial interspecific variation exists in fruit characters such as the persistence of the petals at the apex, mesocarp morphology, endocarp shape, lobing and hardness, and the presence of an apical crown of fibers. Unfortunately, neither Danser (1940) nor Stauffer (1969) provided descriptions for the two species discussed here, thus information from Macklin (2000) and Macklin and Parnell (2002) was consulted. Among the 11 Dufrenoya species, it appears that only D. 82 M.P. Devkota et al. / Flora 215 (2015) 75–83 Fig. 5. Distribution of Dufrenoya platyphylla (dots) and D. granulata (stars) in Nepal. Map from Lillesø et al. (2005), used with permission. platyphylla has an endocarp crowned by fibrovascular fibers (Fig. 4F), sharing this feature with Dendromyza (s. lat., including Cladomyza). These fibers ascend to the apical portion of the mesocarp where they then join fibers that descend to the base of the fruit, running just inside the clusters of brachysclereids (Fig. 4G and H). These fibers have been called “apical tails” in the literature, but are here referred to as a “crown”. The fruit mesocarp was described as having radially aligned, membranous fibers by Danser (1940), a description repeated by Kuijt (2015). The description in Macklin (2000) and Macklin and Parnell (2002) indicate two forms exist among the Dufrenoya species: with long, membranous strands that wrap around the endocarp or with short, membranous strands that are randomly oriented. The latter type supposedly exists in D. platyphylla but was not seen in the fruits examined in this study. It is assumed that the fibrovascular fibers and mesocarp strands function as they do in Dendromyza where, after ingestion and excretion by the foraging bird, these structures help adhere the seed to the host tree branch. 3.5. Generic circumscriptions Given the above results, it is necessary now to critique some recent taxonomic works, specifically their generic circumscriptions. In the treatment of Santalaceae for Flora of China (Xia and Gilbert, 2003), Dufrenoya was included within Dendrotrophe. The rationale for this decision was not explained; however, this action resulted in a combined generic description that did not adequately encompass Dufrenoya. For example, all five species are referred to as “woody vines”, but neither of the two included Dufrenoya species (D. platyphylla and D. granulata) twine, thus they are best described as erect or pendulous shrubs (mistletoes). The number of ovules given is three, but no published data about ovule number in Dufrenoya could be located. Both Danser (1940) and Stauffer (1969) relied heavily upon features of the fruit and seed to differentiate genera in Amphorogynaceae. The description of the fruit and seed by Xia and Gilbert (2003) appears to be a combination of features from Dendrotrophe and Dufrenoya, none of which would appear together in any one species. From the molecular phylogenetic work and morphological data reported here, it is clear that Dufrenoya is distinct from Dendrotrophe. Separate generic status is also supported by morphological characters differences. For example, Dendrotrophe has a fleshy mesocarp and a tuberculate endocarp with chambers at the apex and the base as well as transverse chambers. The mesocarp in Dufrenoya consists of membraneous strands and its endocarp is smooth with chambers but no transverse chambers. 3.6. Distribution of Dufrenoya in Nepal Nepal has traditionally been divided into three physiographic regions (Tarai, Hills and Mountain), but more recently this has been further divided into Terai (13.7% of Nepal, <500 m), Siwaliks (12.8%, 300–1500 m), middle-Mountains (from 29.2%, to 300–3000 m), high Mountains (from 20.3%, to 1000–5500 m) and the high Himalayas (23.9%, 2000–8500 m). Furthermore, ecological zones comparable to Holdridge life zones can be designated: lower tropical, upper tropical, sub-tropical, temperate, subalpine, and trans-Himalayan (Lillesø et al., 2005). Nearly all of the administrative districts of Nepal have a wide range of ecological zones, e.g., Kaski with upper tropical to trans-Himalayan. The printed version of the Flora of Nepal (Hara et al., 1982) lists both D. platyphylla and D. granulata as does the online version of the Annotated Checklist of the Flowering Plants of Nepal (Press et al., 2000). From our herbarium investigations, 12 discrete localities were recorded for D. platyphylla and 8 localities for D. granulata (Table 1, Fig. 5). The distribution of Dufrenoya in Nepal is strongly associated with physiographic region. With the exception of three D. granulata collections, all were located in the middle Mountain region or near its border with the high Mountains region. The Beer (5 November 1975) and Suzuki (23 May 1992) collections were mapped to the high Mountains region; however, in neither case was label information precise plus the locations are very near M.P. Devkota et al. / Flora 215 (2015) 75–83 the border with the middle Mountains region. The Kanai et al. (9 November 1963) collection of D. granulata from Ila Danda, Taplejung District was mapped to the high Himalayan region, but in a narrow river valley at 2300 m. All Dufrenoya collections examined occur in the eastern half of the country, but this may be simply an artifact of collecting. Suitable habitat in the middle Mountains region is present, thus future field work may locate this mistletoe in the mid-Western and far-Western regions. In at least three districts (Kaski, Sankhuwasabha and Taplejung), collections of both D. platyphylla and D. granulata have been made within close proximity, thus it can be deduced that these species are sympatric over a portion of their respective ranges. 3.7. Protection status Relatively few Dufrenoya collections have been made in Nepal and most herbarium specimens are old: the mean year for all collections examined for D. platyphylla is 1950, for D. granulataem > 1975. Most examined collections fall outside of protected areas. The type location for D. platyphylla (Wallich 488) may have been collected within what is currently designated the Shivapuri Nagarjun National Park. In Kaski District, one collection of D. granulata (Dawson 492) and two collections of D. platyphylla were made just south of the border of the Annapurna National Park, thus it is possible these taxa occur within the park confines. It appears that the Kanai et al. collection of D. granulata from Taplejung district occurs within the Kanchenjungha conservation area. The middle Mountain region is undergoing rapid change with the conversion of natural ecosytems into human-dominated landscapes through forestry, farming, and settlement expansion. As an aerial parasite, Dufrenoya is dependent upon intact populations of compatible host trees, thus it is very susceptible to deforestation. Protection of suitable habitats for biodiversity conservation in general and Dufrenoya in particular must include the East Himalayan Oak-Laurel forests in temperate zones and the Schima-Castanopsis forest in sub-tropical zones. Because many regions of Nepal have not received floristic inventories, it is difficult to estimate the number and size of Dufrenoya populations within the country. But because of modifications of the middle Mountain region landscapes, fragmentation of mistletoe populations is likely. Thus, we estimate that both species of Dufrenoya should receive the IUCN classification of vulnerable. 4. Conclusions Two well-defined species of Dufrenoya, D. granulata and D. platyphylla, occur in Nepal, mostly restricted to the middle Mountain region in the eastern half of the country. The genus is important because it is on a clade representing one of the five cases of independent evolution of the mistletoe habit. The nomenclatural history of Dufrenoya is complex, complicated by varying generic concepts and incomplete taxonomic descriptions. Previous molecular work and the ITS phylogeny reported here clearly show that Dufrenoya is distinct from Dendrotrophe and Dendromyza and is most closely related to another mistletoe, Phacellaria. Although, the genus is generally reported to be dioecious, evidence exists that more complex sexual systems may be in place. Further study is required to determine the sexual systems and floral visitors/pollinators occurring in populations of the 11 species in the genus. The apical crown of fibrovascular fibers is known only from D. platyphylla, a feature shared with Dendromyza. The functional role such morphological and anatomical structures play requires elucidation, as well as field studies to document details of seed dispersal. We estimate that both 83 species of Dufrenoya in Nepal should receive the IUCN classification of vulnerable because of human alteration of natural ecosystems where its host trees occur. Acknowledgements We are grateful to the curators of the following herbaria who allowed access to their specimens: E, BM, K, KATH, and TI. References APG III, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants. Bot. J. Linn. Soc. 161, 105–121. Ashworth, V.E.T.M., 2000. Phylogenetic relationships in Phoradendreae (Viscaceae) inferred from three regions of the nuclear ribosomal cistron. I. Major lineages and paraphyly of Phoradendron. Syst. Bot. 25, 349–370. Bista, M.S., Adhikari, M.K., Rajbhandari, K.R., 2001. Flowering Plants of Nepal (Phanerogams), Bulletin of Department of Plant Resources. National Herbarium and Plant Laboratories. Department of Plant Resources, Ministry of forest and soil Conservation/HMGN, Kathmandu, Nepal. Chatin, G.A., 1860. Anatomie Comparée des végétaux — Formation du genre Dufrenoya et rétablissement d’un genre Sphaerocarya. Comptes Rendus Hebdomadaires des Séances de l’Académie des Sciences. Paris, Mallet-Bachelier, Imprimeur-Libraire 51, 657–659. Danser, B.H., 1940. On some genera of Santalaceae Osyrideae from the Malay Archipelago mainly from New Guinea. Nova Guinea N.S. 4, 133–150. Danser, B.H., 1955. Supplementary notes on the Santalaceous genera Dendromyza and Cladomyza. Nova Guinea N.S. 6, 261–277. Der, J.P., Nickrent, D.L., 2008. A molecular phylogeny of Santalaceae (Santalales). Syst. Bot. 33, 107–116. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11–15. Ewart, M.F., 1892. On the staminal hairs of Thesium. Ann. Bot. 6, 271–290. Hara, H., Chater, A.O., Williams, L.H.J., 1982. An Enumeration of the Flowering Plants of Nepal. British Museum (Natural History), London. Hara, H., Stearn, W.T., Williams, L.H.J., 1978. An Enumeration of the Flowering Plants of Nepal. Trustees of British Museum (Natural History), London. Hara, H., Williams, L.H.J., 1979. An Enumeration of the Flowering Plants of Nepal. Trustees of British Museum (Natural History), London. Kuijt, J., 2015. Santalales. In: Kubitzki, K. (Ed.), The Families and Genera of Vascular Plants. XII Flowering Plants: Eudicots Santalales, Balanophorales. Springer International Publishing, Cham, Switzerland, pp. 1–189. Lillesø, J.-P.B., Shrestha, T.B., Dhakal, L.P., Nayaju, R.P., Shrestha, R., 2005. The Map of Potential Vegetation of Nepal: a Forestry/Agro-Ecological/Biodiversity Classification System. Center for Skov, Landskab og Planlægning/Københavns Universitet, Hørsholm (Development and Environment; No. 2/2005). Macklin, J., 2000. A Systematic Revision of the Santalaceae R. Br. of Southeast Asia, Botany. Trinity College, Dublin. Macklin, J., Parnell, J., 2002. An account of the Santalaceae of Thailand. Thai For. Bull. 30, 75–115. Molvray, M., Kores, P.J., Chase, M.W., 1999. Phylogenetic relationships within Korthalsella (Viscaceae) based on nuclear ITS and plastid trnL-F sequence data. Am. J. Bot. 86, 249–260. Nickrent, D.L., García, M.A., Martín, M.P., Mathiasen, R.L., 2004. A phylogeny of all species of Arceuthobium (Viscaceae) using nuclear and chloroplast DNA sequences. Am. J. Bot. 91, 125–138. Nickrent, D.L., Malécot, V., Vidal-Russell, R., Der, J.P., 2010. A revised classification of Santalales. Taxon 59, 538–558. Press, J.R., Shrestha, K.K., Sutton, D.A., 2000. Annotated Checklist of the Flowering Plants of Nepal. The Natural History Museum, London. Rambaut, A., 2007. Se-Al Sequence Alignment Editor, 2.0 a11 ed. Department of Zoology. University of Oxford, Oxford, UK. Rao, L.N., 1942. Studies in the Santalaceae. Ann. Bot. 6, 151–175. Stauffer, H.U., 1969. Santalales-Studien X. Amphorogyneae eine Neue Tribus der Santalaceae. Vierteljahrsschrift Naturf. Ges. Zürich 114, 49–76. Sun, Y., Skinner, D.Z., Liang, G.H., Hulbert, S.H., 1994. Phylogenetic analysis of Sorghum and related taxa using intemal transcribed spacers of nuclear ribosomal DNA. Theor. Appl. Genet. 89, 26–32. Swofford, D.L., 2002. PAUP*: phylogenetic analysis using parsimony (* and other methods), 4.0. b10 ed. Sinauer Associates, Sunderland, MA. Vidal-Russell, R., Nickrent, D.L., 2008. The first mistletoes: origins of aerial parasitism in Santalales. Mol. Phyl. Evol. 47, 523–527. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and Direct Sequencing of Fungal Ribosomal Genes for Phylogenetics. Academic Press, San Diego, CA, Chapter 38. Xia, N.H., Gilbert, M.G., 2003. Santalaceae, Flora China. Science Press & Missouri Botanical Garden Press, Beijing & St. Louis, pp. 208–219. Zuber, D., 2004. Biological flora of central Europe: Viscum album L. Flora 199, 181–203.