INTERNATIONAL JOURNAL OF CONSERVATION SCIENCE ISSN: 2067-533X Volume 10, Issue 4, October-December: 721-732 www.ijcs.uaic.ro STUDIES ON THONNINGIA SANGUINEA VAHL. (BALANOPHORACEAE) IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE Odoligie IMARHIAGBE1,*, Emmanuel Izaka AIGBOKHAN2 1 Department of Biological Sciences, Edo University Iyamho, Nigeria. Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria 2 Abstract Studies on the host-parasite relationship are intrinsically linked with the nature of parasitic plants. No such records have been recorded for Thonningia sanguinea (Balanophoraceae), a rare, cryptic, obligate holoparasitic plant endemic to tropical Africa. This study reports on its host range and preference in Southern Nigeria. Its presence and current distribution ranges were plotted from reconnaissance surveys conducted across selected forested areas in Southern Nigeria, comprising, national parks, forest reserves, and community-managed forests. To detect specific hosts, soil excavation leading from parasite to host was conducted. The results showed a broad host range with a special preference for native trees and members of the Euphorbiaceae and Urticaceae. Among the common host trees were: Guarea cedrata (Meliaceae), Lophira alata (Ochanaceae), Musanga cecropiodes (Urticaceae), Myrianthus arboreus (Urticaceae), and Ricinodendron heudelotii (Euphorbiaceae). Only Hevea brasiliensis and Theobroma cacao were of exotic origin. Lophira alata was the most susceptible infected host plant with a percentage infestation rate of 28.77, while Musanga cecropiodes had the highest percentage occurrence as host with 31.57 %. This finding could have significant implications on the conservation status of the plant, which accounting for current IUCN status is Not Evaluated (NE). In situ conservation is however recommended. Keywords: Parasitic plant; Cryptic; Host range; Conservation; Thonningia sanguinea Introduction The family Balanophoraceae is mostly herbaceous, perennial root parasites, arising from underground tubers attached to the roots of the host [1]. Worldwide, there are 18 representative genera and 44 species, mostly tropical [2-3]. Some notable genera such as Corynaea, Helosis, Langsdorffia, Lophophytum, Ombrophytum and Scybalium have a neotropical distribution, while Thonningia, Sarcophyte, Dactylanthus are restricted to paleotropical region. However, Thonningia sanguinea is the only example of this family that is common in West Africa. Members of this family are normally found in moist inland forests growing on tree roots and have an above ground inflorescence with the overall appearance, similar to that of a fungus, composed of numerous minute flowers. The inflorescences develop inside the tuberous underground part of the plant, before rupturing and surfacing. Several ecological studies carried out on common genera, such as Balanophora [4-5] Corynaea crassa [6], Ombrophytum [7], Langsdorffia [8] and the New Zealand endemic Dactylanthus [9] have attested to the catholic host utilization of the family. Due to their * Corresponding author: imarhiagbe.odoligie@edouniversity.edu.ng O. IMARHIAGBE and E.I. AIGBOKHAN holoparasitic nature, the distribution of its members is largely limited by host availability, host quality, host resistance to parasitism, and parasite preference [10-13]. Therefore, a fundamental aspect of the ecology of these species is to understanding their host-parasite interaction, which also is a prerequisite step to their conservation [8, 14]. Thonningia sanguinea (Balanophoraceae) is a cryptic obligate holoparasitic plant endemic to tropical Africa. It is a fleshy dioecious herb growing from a branching, yellow underground tuber which extends horizontally up to 10 or 15 centimeters through the soil with which it forms bulb-like swellings at the point where it attaches to the roots of its host plants [15]. Records from literature and databases revealed little available records on T. sanguinea. Apart from narratives on its habit [15] and bioassay on its medicinal properties [16-18], very few studies exist on the ecology of T. sanguinea. Therefore, the present study was conducted based on the following objectives: (i) delineate the scope of T. sanguinea (ii) document its host species and (iii) identify its host preference in Southern Nigeria. Materials and Methods Population Location Due to the cryptic and non-random nature of Thonningia sanguinea distribution within the forest environment, T. sanguinea populations were located using the following: meander searches, published materials, and personal communications. Study Sites A total of 24 locations habouring T. sanguinea were randomly selected from eight (8) Okomu National Park, five Cross River National Park, one Akure Ofosu Forest Reserve, two International Institute of Tropical Agriculture (IITA) Forest Reserve, one Idanre Forest Reserve, two Oba Hills Forest Reserve, four Community Managed-Forests (namely: Okokhuo, Ehor Nu Wire, Okuor and Iyanomo Rubber Plantation Forest (Fig. 1). Fig. 1. Map of Nigeria (inset) showing study sites in selected southern states Sampling The distribution of T. sanguinea within forested location was assessed using a stratified sampling method to mark and quantify the number of populations present in each sampling location. On host–parasite distance determined from the previous preliminary study shows that 722 INT J CONSERV SCI 10, 4, 2019: 721-732 STUDIES ON THONNINGIA SANGUINEA VAHL. IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE host species do not extend beyond an average of 3.4 meters away from the parasite. Hence, a 10×10m quadrat was adopted to accommodate host trees present [19]. Host species To identify the host plants, Thonningia haustoria were excavated manually using cutlass, trowels and soil scoops following the procedure reported by M.A. Marvier and D.L. Smith [14]; M.A. Piehl [20]; T.I. Chuang and L.R. Heckard [21]; C.C. Gibson and A.R. Watkinson [22], and the host-plant roots were traced to the main stem and from which a determination was made using: floras [23], manuals [24-27] and checklists [28]. Plants whose identity presented some challenges were pressed and preserved and subsequently determined using a standard taxonomic key as outlined by W.L. Applequist [29]. Table 1. Host species and Sites Habouring Thonnimgia sanguinea in Southern Nigeria Study Location Okomu National Park Cross River National Park Idanre Forest Reserves IITA Forest Reserves Ofosu Forest Reserves Oba hills forest Reserves Community forest Plantation Forest Sites Host species Coordinates Altitude (m) Compartment 33 Compartment 52 Compartment 53 Compartment 53 Compartment 54 Compartment 55 Buffer zone Buffer zone Rock formation Zone Forest interior Rock surface Cocoa plantation Forest interior Cocoa plantation Cocoa plantation Ehor Nu Wire Forest Okokhuo Forest Okour Forest Iyanomo Forest Guarea cedreta Myrianthus arboreus Guarea cedreta Myrianthus arboreus Musanga cecropiodes Ricinodendron heudelotii Host unknown Lophira alata Musanga cecropiodes Lophira alata Host unknown Theobroma cacao Host unknown Musanga cecropiodes Host unknown Hevea brasiliensis Hevea brasiliensis Hevea brasiliensis Hevea brasiliensis 06°24.113” 005° 19.440”E 06°21.381” 005°19.889” E 06°20.937” 005°20.685” E 06°20.135” 005°20.470” E 06°21.062” 005°21.398” E 06°21.656” 005°21.587” E 05°21.863ʹʹ 008°26.438ʹʹ E 05°22.172” 008° 26.182” E 05°21.863” 008° 26.438” E 05°21.925” 008°26.350” E 05°21.863” 008°26.438” E 07°01.954” 005°09.868” E 07°29.820” 003053.530” E 06°43.278” 005007.852” E 07°45.275” 004°07.752” E 06°18.342” 005° 48.598” E 06°34.909” 005° 36.415” E 06°11.962” 006° 04.928” E 06°09.746” 005°34.898” E 5.4 77.5 102.6 94.8 59.6 10.2 139.2 167.6 139.2 123.6 139.5 196.6 214.5 188.6 253.3 99.1 25.3 148.6 35.3 Degree of infestation The hastorium of Thonningia sanguinea possesses several ramifications, growing in a rather irregular horizontal manner below the ground which makes it difficult to quantify the number of parasites attached to a host. The number of inflorescence attached to each host was recorded and used to estimate the degree of infestation following the procedures of C.J. Thorogood, S.J. Hiscock [30]. Statistical Analysis Data collected on the occurrence of host plants in study sites and the degree of infestation on different host species were presented in a graphical format. All statistics were performed using SPSS ver. 23. Results and Discussion The cryptic nature of T. sanguinea and the fact that it has attracted little research attention makes it imperative to investigate its host range and preference as a fundamental step towards developing a conservation strategy for these understudied species. The majority of studies concerning host range in root parasites have concentrated mostly on examination of parasite performance in pots with different host species as an alternative to excavation study [31]. However, pot-based studies are not appropriate for a full understanding of the host range of a parasitic plant. According to C.J. Thorogood et al. [32], pot-based studies may result in an http://www.ijcs.uaic.ro 723 O. IMARHIAGBE and E.I. AIGBOKHAN ambiguous predictive pattern of host use in the wild. To arrive at a more encyclopedic host list that supports the populations of T. sanguinea in Southern Nigeria, the present study investigated the host range of T. sanguinea in its habitat which consists of natural and plantation forests. The result from the study revealed that Thonningia sanguinea individuals parasitized six host species, of different angiosperm families (Table. 1). This finding suggests a broad host range for T. sanguinea with members of the Euphorbiaceae and Urticaceae being the most susceptible. Except for the few exotic ones (Hevea brasiliensis and Theobroma cacao), the host species of T. sanguinea in Southern Nigeria were native, they include Ricinodendron heudelotii (Euphorbiaceae), Myrianthus arboreus (Urticaceae), Musanga cecropiodes (Urticaceae), Lophira alata (Ochanaceae), and Guarea cedreta (Meliaceae). The implication of Thonningia sanguinea embraces host species from different taxonomic families, suggest that the choice of host type could be based on the quality of hosts in the local plant community of its distribution range in Southern Nigeria. The broad host range occurrence had previously been observed in other members of Balanophoraceae, such as Ombrophytum subterraneum, which parasitizes 10 different hosts distributed in four angiosperm families and also in Langsdorffia hypogaea, which parasitizes six hosts in five different angiosperm families; both in Brazil [2]. But unlike L. hypogaea with which showed no specificity in terms of the life-form of the host species, parasitizing species of lianas, tree, and even cactus [8], host species of T. sanguinea were all long-lived mature perennials, trees and as such are capable of a long term support for the parasite (Table 1). According to M.A. Marvier and D.L. Smith [14], the majorities of parasitic plants have opted for a mature perennial host for successful growth and in so doing, affords them the opportunity of utilizing the advantages of an already established deep root system of the host. The generalist phenomenon in T. sanguinea as regard host use further corroborates the previously held notion that members of Balanophoraceae are not specific to a single host and are mostly catholic in their choice of host. They may have evolved this broad generalist strategy to increase their survival chances since they are obligate parasites that cannot exist in the absence of a host. D.L. Nickrent [33], suggested that the lack of host specialization in members of Balanophoraceae could be the reason for their occurrence in tropical regions where the forests are characterized by a high potential host diversity. Table 2. Phytogeographical status and life-form of the host plant species of Thonningia sanguinea in Southern Nigeria Host species Family Gurea cedrata (A. Chev.) Pellegrin Hevea brasiliensis Mull.Arg. Lophira alata Banks ex Gaertn. Musanga cecropiodes R.Br. & Tedlie Myrianthus arboreus P. Beauv. Ricinodendron heudelotii (Baill.) Heckel Theobroma cacao L. Meliaceae Euphorbiaceae Ochnaceae Urticaceae Urticaceae Euphorbiaceae Malvaceae Phytogeographical status Life form Forest type Native Exotic Native Native Native Native Exotic Tree Tree Tree Tree Tree Tree Tree Secondary forest Plantation forest Secondary forest Secondary forest Secondary forest Secondary forest Plantation forest According to P.R. Atsatt [34], parasitic plants can be able to attack many host species throughout its range, whereas the local population of parasite remains quite host-specific. In the present study, although T. sanguinea utilizes different host species within the scope of its distribution in southern Nigeria, However, we did not observe two or more host species for a local population of the parasite. R.N. Glover et al. [35] reveal that different host species make different kinds of nutritional contribution to sustaining the general population of the parasite. This further suggests that T. sanguinea seems sufficiently contented with its choice of host and as such precludes the chances of seeking another within a local environment. Aside from the definitive host species recorded certain species like Albizia lebbeck, Allanblankia floribunda, Barteria fistolosa, Cleistopholis patens, Funtumia elastica, Harugana madagascariensis, Strombosia grandifolia, and Voacanga africana were also observed to show 724 INT J CONSERV SCI 10, 4, 2019: 721-732 STUDIES ON THONNINGIA SANGUINEA VAHL. IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE a high frequency of association with T. sangunea. These species observed to show regular association with T. sanguinea could be regarded as potential hosts (Fig. 2). Fig. 2. Tree species showing percentage frequencies of association with T. sanguinea sites in Southern Nigeria The capacity of T. sanguinea to utilize exotic species as a host demonstrates the tendency to interact with non-native species of its distribution range. Such an association may have occurred through the cultivation of such exotic species on the site previously inhabited by T. sanguinea thereby resulting in re-establishment from seeds remaining in the soil after the native forest was cleared off. The likelihood of seed dispersal from adjacent close native forest to areas now occupied by exotic host species such as Hevea brasiliensis and Theobroma cacao can also be responsible for such an association. Previous parasitic host researchers have revealed that parasite performance varies greatly with the availability of different host species and the relative importance of particular host http://www.ijcs.uaic.ro 725 O. IMARHIAGBE and E.I. AIGBOKHAN species may not immediately be obvious [14]. Several factors determine the suitability of hosts, which further increases the frequency of infestation on host vis-à-vis host preferences. Some of these factors include the probability of contracting and successfully attacking a host, the quality of the host and host duration. In the present study, Lophira alata with a percentage infestation of 28.77% was the most susceptible host plant. The level of susceptibility for Myrianthus arboreus, Ricinodendron heudelotii, Musanga cecropiodes, Hevea brasiliensis, Guarea cedreta, Theobroma cacao were 21.57%, 14.89%, 10.68%, 8.88%, 8.73%, 6.46% respectively (Fig. 3). Although a number of reasons might be responsible for the unequal percentage of infestation on various hosts, we believe insights into the biochemical assessments of host plants could help explain their palatability and compatibility. The analysis of the percentage occurrence of host species on the different sites shows Musanga cecropiodes had the highest occurrence with 31.57 % followed by Hevea brasiliensis with 26.31 %; others had a varying degree as follows: Myrianthus arboreus ≥ Lophira alata ≥ Theobroma cacao > Ricinodendron heudelotii > Guarea cedreta (Fig. 4). This finding shows Musanga cecropiodes represents a well distributed host species in forested areas of Southern Nigeria, as inferred by its high percentage frequency of occurrence as host. Fig. 3. Percentage infestation rates of Thonningia sanguinea on different host species Fig. 4. Percentage occurrence of host plants in surveying sites 726 INT J CONSERV SCI 10, 4, 2019: 721-732 STUDIES ON THONNINGIA SANGUINEA VAHL. IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE Implication for conservation Conservation concerns about parasitic plants pose a challenge that requires us to view parasitism from an uncommon perspective, therefore, we must cast off our usual attitude regarding parasites and determine how best to increase their abundance and reproductive success. The host population has serious implications for holoparasitic plant species due to their total dependence on the host. M.A. Marvier and D.L. Smith [14] reported that a good conservation and restoration practice for parasitic plants must necessitate the management of thoughtfully selected host populations. The present study has identified T. sanguinea host plants to include both native and exotic tree species (Guarea cedrata Hevea brasiliensis Lophira alata Musanga cecropiodes Myrianthus arboreus, Ricinodendron heudelotii, and Theobroma cacao). While the native host species support the majority of Thonningia sanguinea populations in protected sites (National Parks and Forest Reserves). The exotic host supports the population of T. sanguinea in community forests (unprotected sites) (Fig. 5). Therefore, the continued survival of T. sanguinea in these habitats is largely dependent on the sustainable protection of the host population. In Nigeria, National Parks are to some extent protected with little or no habitat destruction; however, Forest Reserves appear to have been intensely invaded by the local people living around it. Most of the Forest Reserves like Idanre and Ofosu lack proper protective boundaries, thereby allowing access to it. Fig. 5. Incidence of Thonningia sanguinea in protected and unprotected sites of Southern Nigeria (The unprotected sites consist of: Ehor Nu Wire forest, Iyanomo forest, Okour forest, Okokhuo Forest, while the protected sites were: Okomu National Park, Cross River National Park, Idanre Forest Reserves, Ofosu forest Reserve, Oba Hills Forest Reserves, and IITA Forest Reserves) The IUCN red list category for T. sanguinea is “Not Evaluated” (NE). Consequently, concerted efforts needed to conserve T. sanguinea populations in Southern Nigeria must be put in place. At present, the seeds are quite rare and the mechanism of domestication is still elusive among scientists. Therefore In situ conservation is best recommended to sustain the current wild populations of the plant. Conclusion The relevance of host plant species to holoparasitic plants cannot be overemphasized. They serve as “lynchpins” for the continued existence of the parasite. The present study has demonstrated that T. sanguinea uses a wide range of host species from different angiosperm families, some of which are more heavily infested than others. http://www.ijcs.uaic.ro 727 O. IMARHIAGBE and E.I. AIGBOKHAN The key conservation recommendation is to maintain the forest margin paths, such as forest trails in National Parks and Forest Reserves where T. sanguinea seem to be prevalent. And also, protect the populations of Hevea brasilienesis and Theobroma cacao serving as host in community forests around Southern Nigeria. Acknowledgement The authors are grateful to the managements of the various National Parks and Forest Reserves for granting permission to carry out this research. This work was completely funded by the TETFUND 2017-2018 (Batch 12th) Research Project (RP) Intervention. References [1] B.L. Stannard, Balanophoraceae, Flora Zambesiaca, 9(3), 2006, pp. 249 – 251. [2] B. Hansen, Balanophoraceae, Flora Neotropical, 23, 1980, pp. 1 - 80. [3] M.J.M. 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Ecroyd, The ecology of Dactylanthus taylorii and threats to its survival, New Zealand Journal of Ecology, 20(1), 1996, pp 81-100. [10] J.G. Garcia-Franco, V. Rico-Gray, Distribution and host specificity in the holoparasite Bdallophyton bambusarum (Rafflesiaceae) in a tropical deciduous forest in Veracruz, Mexico, Biotropica, 28 (4), 1996, pp. 759-762. [11] D.A. Norton, M.A. Carpenter, Mistletoes as parasites: host specificity and speciation, Trends in Ecology and Evolution, 13(3), 1998, pp. 101-105. [12] D.A. Norton, P.J. De Lange, Host specificity in parasitic mistletoes (Loranthaceae) in New Zealand, Functional Ecology, 13(4), 1999, pp. 552-559. [13] M.C. Press, G.K. Phoenix, Impacts of parasitic plants on natural communities, New Phytologist, 166(3), 2005, pp. 737-751. [14] M.A. Marvier, D.L. Smith, Conservation implication of host use for rare parasitic plants, Conservation Biology, 11, 1997, pp. 839-848 [15] V.O. Otoide, Thonningia sanguinea- a new parasite on rubber roots, Tropical Pest Management, 28(2), 1982, pp. 186-188. [16] M.A. Gyamfi, M. Yonamine, Y. Aniya, Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries, General Pharmacology, 32(6), 1999, pp. 661-667 [17] B. Ouattara, A. Coulibaly, A. M. Kra, F. Guede-Guina, Assessment of antifungal activity of an African medicinal herb Thonningia sanguinea against Cryptococcus neoformans, World Applied Sciences Journal, 3(2), 2008, pp. 191-194. 728 INT J CONSERV SCI 10, 4, 2019: 721-732 STUDIES ON THONNINGIA SANGUINEA VAHL. IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE [18] A.K. Thomford, R.F.A. Abdelhameed, K. Yamada, Chemical studies on the parasitic plant Thonningia sanguinea Vahl, Royal Society of Chemistry Advances, 8, 2018, pp. 21002– 21011 [19] O.F. Agianaku, E.I. Aigbokhan, Flora of Ogba Zoo and Nature Park, Benin City, Nigeria: I. Vegetation Structure, Nigerian Journal of Botany, 31(1), 2018. [20] M.A. Piehl, Mode of attachment, haustorium structure and hosts of Pedicularis Canadensis, American Journal of Botany, 50, 1963, pp. 935-942. [21] T.I. Chuang, L.R. Heckard, Observations of root-parasitism in Cordylanthus (Scrophulariceae), American Journal of Botany 58, 1971, pp. 218-228. [22] C.C. Gibson, A.R. Watkinson, The host range and selectivity of a parasitic plant: Rhinanthus minor L., Oecologia, 78, 1987, pp. 401- 406. [23] J. Hutchinson, J.M. Dalziel, Flora of West Tropical Africa, Second Edition. Crown Agents for Overseas Governments and Administrations, London, 1968, p. 276. [24] R.W. J. Keay, Trees of Nigeria, Clarendon Press, Oxford, 1989, p. 476. [25] M. Arbonnier, Trees, Shrubs and Lianas of West African Dry Zones. Margraf Publishers GMBH. Germany, 2004, p. 572. [26] M. Steentoft, Flowering Plants in West Africa. Cambridge University Press. United Kingdom, 2008, p. 350. [27] I.O. Akobundu, F. Ekeleme, C.W. Agyakwa, C.A. Ogazie, A Handbook of West African Weeds, International Institute of Tropical Agriculture, Ibadan, 2016, p. 381. [28] E.I. Aigbokhan, Annotated Checklist of Vascular Plants of Southern Nigeria-A Quick Reference Guide to the Vascular Plants of Southern Nigeria: A Systematic Approach, Uniben Press. Benin City, 2014, 346p. [29] W.L. Applequist, Taxonomic Keys. Plant Sciences, 2001. [Accesed on 26 June, 2017]. [30] C.J. Thorogood, S.J. Hiscock, Host specificity in the parasitic plant Cytinus hypocistis, Research Letters in Ecology, 84234, 2007, pp. 1-4. [31] V.M. Malcolm, Root parasitism of Castilleja coccinea, Ecology, 47, 1966, pp. 179-186. [32] C.J. Thorogood, F.I. Rumsey, S.J. Hiscock, Host specific races in holoparasitic Orobanche minor: implications for speciation in parasitic plants, Annals of Botany, 103, 2009, pp. 1005-1014. [33] D.L. Nickrent, Parasitic Plants in the World, In: J.A. Lopez-Saez, P. Catalan, and L. Saez, (eds.), Parasitic Plants of the Iberian Peninsula and the Balearic Islands, Mundi-Press Books, Madrid, 2002, pp. 7-27. [34] P.R. Atsatt, Host-parasite interactions in higher Plants, In: P.S. Lange, C.B. Nobel, and H. Z. Osmond (eds.), Encyclopedia of Plant Physiology New Series. Springer-Verlag, Berlin, 1983, pp. 519-535. [35] R.N. Govier, M.D. Nelson, J.S. Pate, Hemiparasitic nutrition in angiosperms. I. The transfer of organic compounds from host to Odontites verna (Bell.) Dum. (Scrophulariaceae), New Phytologist, 66, 1967, pp. 285-297. _____________________________________ Received: March 01, 2019 Accepted: October 10, 2019 http://www.ijcs.uaic.ro 729 O. IMARHIAGBE and E.I. AIGBOKHAN ANNEX 1. Thonningia sanguinea showing (A) Female inflorescence (B) Male inflorescence (C) Male and female inflorescence head in close proximity (D) Fruiting head and (E) & (E) Growth pattern 730 INT J CONSERV SCI 10, 4, 2019: 721-732 STUDIES ON THONNINGIA SANGUINEA VAHL. IN SOUTHERN NIGERIA. RANGE AND HOST PREFERENCE ANNEX 2. Some host species of Thonningia sanguinea in Southern Nigeria (A): Ricinodendron heudelotii (B) Lophira alata (C) Musanga ceropiodes (D) Myrianthus arboreus (E) Theobroma cacao (F) Hevea brasiliensis. http://www.ijcs.uaic.ro 731 O. IMARHIAGBE and E.I. AIGBOKHAN ANNEX 3. Thonningia sanguinea Haustorium with multiple host root attachments (A); Haustorium attachment in Thonningia (B, C & D; (E) Thonningia sangunnea preference for Richinodendron as against Trichilia monadelpha 732 INT J CONSERV SCI 10, 4, 2019: 721-732