﻿An account of the genus Cistanche (Orobanchaceae) in Iraq and taxonomic considerations in the Middle East

﻿Abstract Species limits in the genus Cistanche are poorly understood, despite the plants’ long history of use in traditional herbal medicine and food across their range. Here we present a taxonomic account for the genus Cistanche in Iraq, where several taxa have been reported, most of them doubtfully. Using herbarium specimens, images of living material, and taxonomic literature, we found evidence of only one species occurring with certainty in Iraq: Cistanchetubulosa. We found no evidence for the occurrence of other Cistanche species in Iraq, including a putative new entity reported for the region. Our work highlights inconsistencies in the literature, and underscores the importance of examining multiple stable characters for delimiting species in the genus Cistanche.


Introduction
Iraq has a rich flora with an estimated 3300 species owing to a convergence of phytogeographic regions and varied climate and topography (Zohary 1973;Ghazanfar and McDaniel 2016).Until the mid-twentieth century, there were no checklists or Floras for this young state (Frodin 2001;Ghazanfar and McDaniel 2016).In the 1950s, the first national checklist, The Flora of Iraq and its Phytogeographical Subdivision, was written by Michael Zohary (Frodin 2001;Ghazanfar and McDaniel 2016).In 1964 Karl-Heinz Rechinger published the Flora of Lowland Iraq (Rechinger 1964a) with contributions by multiple authors, including a treatment for the Orobanchaceae by H. Schiman-Czeika.A year later, the Flora of Iraq began as a collaborative project between the Royal Botanic Gardens, Kew and the Ministry of Agriculture, Baghdad (Frodin 2001;Ghazanfar and McDaniel 2016).From this project, volumes 1, 2 and 3, and volume 4 parts 1 & 2, 8 and 9 were published; volumes 5, 6 and 7 remained unpublished due to political instability in the region, and the project was suspended in the 1980s (Guest and Townsend 1966;Townsend andGuest 1966-1985;Frodin 2001;Ghazanfar and McDaniel 2016).Then in the 2010s, the Flora of Iraq project resumed as a collaboration between the Royal Botanic Gardens, Kew and the Ministry of Agriculture, Baghdad, in 2013, Vol. 5(2) was published and vol.5(1) is in press.Volumes 6 and 7 which cover around 900 species are in progress (Ghazanfar and McDaniel 2016).The family Orobanchaceae has been written for this ambitious project but remains, as yet, unpublished.
The genus Cistanche Hoffmanns.& Link (family Orobanchaceae), was first identified officially in 1799 by Ventenat under the name Orobanchoideae.A decade later, the genus Cistanche was described by Hoffmannsegg and Link (1813).The latest monograph for the family Orobanchaceae was published in 1930 by Beck-Mannagetta (Beck-Mannagetta 1930).He divided Cistanche species into four sections based on calyx and bracteole morphology; however the first comprehensive phylogeny for the genus Cistanche revealed that none of these sections are monophyletic, with the exception of C. sect.Subcistanche (Ataei et al. 2020).Moreover, this phylogeny revealed that Cistanche species form four well-supported, geographically differentiated clades which they described as the Northwest African Clade, Southwest Asian Clade, Widespread Clade and East Asian Clade.The East Asian Clade is the only clade that corresponds to a previously recognized taxonomic section (C.sect.Subcistanche).Despite progress in understanding the evolutionary relationships in the genus, a well-sampled phylogeny substantiated with detailed morphological and ecological data are absent, and species limits remain confused and uncertain.
Cistanche is a holoparasite that lacks vegetative traits traditionally used in taxonomy, including functional leaves and roots.The poor condition of herbarium specimens -particularly type specimens -has generated confusion in identification.Here we examine herbarium specimens, images of living material, and taxonomic literature to produce the first robust review of the genus Cistanche in Iraq, which will inform the treatment for the Flora, and other treatments for the genus in the Middle East.

Study species
We reviewed the names used for Cistanche in Iraq and neighbouring countries.The sources that were used to identify species of Cistanche putatively in Iraq, and the species in their accounts, were as follows: Treatments including Iraq  (Karim 1978) cites only C. tubulosa.Karim (1978) was aware of seven plants and cited three host species.4. In an unpublished PhD thesis that included a monographic treatment of genus Cistanche, Ataei (2017) cited two species in the exsiccatae for Iraqi specimens: C. tubulosa and C. flava.She referred to the C. salsa specimen cited by Rechinger (1964a), considering it to be a misidentified specimen of C. ambigua (Bunge) Beck.In the treatment she also referred to an, as yet, unpublished species found in Iraq, but no specimen from Iraq was cited.

Treatments of neighbouring countries
1.The Flora of Syria, Palestine and Sinai (Post 1932)

Distribution mapping
Google Earth was used to create coordinate assumptions for those specimens for which true coordinates were unavailable (Fig. 1).Coordinates were exported to QGIS 3.4 with layers from the Natural Earth Quick Start Kit.Host species were recorded from specimen labels or based on identifications of host plant material mounted on the same sheet as the specimen.In the absence of excavation of host-parasite connections, the identity of the host was considered tentative (the parasite can appear some distance above ground from the host plant).Finally, we considered C. chabaharensis, an as yet unpublished name referred to in the thesis of Ataei (2017) (Fig. 2B).This description placed focus on the glabrous, acute anthers; the key also described bract and bracteoles to possess non-sinuate margins, yellow corolla, and anther filaments glabrous at the base.However, on close examination of specimens cited by Ataei (2017) from Oman (McLeish, E00121976 E) and Iraq (Barkley & Abbas-Al-Ani, 6499 K), it was apparent both had woolly anthers that could not be distinguished from anthers of C. tubulosa, and that the bracts and bracteoles were sinuate.We consider it doubtful that this unpublished name represents a new entity found in Iraq; rather we believe this to be an entity that falls within the bounds of variability in C. tubulosa s.l.In summary, only three species warranted detailed investigation for the region in question: C. tubulosa, C. salsa and C. flava (Table 1; Fig. 3).

Morphology and evolutionary relationships
Here we consider the three taxa identified to potentially co-occur in the region.Cistanche salsa can be readily differentiated from C. flava and C. tubulosa by its hairiness; the latter two species are glabrous (see key).Cistanche flava is differentiated from C. tubulosa by its scarious, conspicuously long bracts which are twice as long as the calyx or even longer (Table 1; Figs 3A, D, 4C,  D).Sánchez Pedraja et al. (2016) consider C. flava to be a synonym of C. tubulosa, however the distinct bract morphology is apparent both in living and dried material.Detailed, well-sampled investigations of both taxa are absent, and their distinction remains open to question; however recent molecular work provisionally supports their separation (Ataei et al. 2020).
The name Cistanche tubulosa s.l. is used from Africa and the Middle East to South and Central Asia and China, however as currently applied, the name refers to a widely distributed, polyphyletic group of plants (Aldughayman et al. 2024).In the most complete phylogeny (Ataei et al. 2020), specimens identified as C. tubulosa (or aff.C. tubulosa) were placed in a 'widespread clade', but four specimens identified as aff.C. tubulosa were nested in a separate clade sister to C. flava, along with other plants identified as C. senegalensis (an entity considered by Beck-Mannagetta to be closely related to C. tubulosa, but differentiated on the basis of subequal lower corolla lobes, oblong bracts and the absence of purple pigmentation).Additionally, two specimens identified as C. tubulosa are in a subclade which otherwise includes the Central Asian species, C. laxiflora.The type specimen of C. tubulosa is lost, and the correct application of the name C. tubulosa is yet to be clarified although it does seem to be misapplied to specimens in the C. laxiflora clade (Ataei et al. 2020;Aldughayman et al. 2024).Recently, a specimen collected from South Sinai near the type locality was designated a neotype (Aldughayman et al. 2024).This neotypification, alongside further phylogenetic work, is necessary to re-evaluate whether the name Cistanche tubulosa is a synonym for the name C. tinctoria, as has been proposed by Moreno Moral et al. (2017), and also to confirm whether the name C. tubulosa is the correct name for any Iraqi entity.To avoid compounding confusion, here we use the name C. tubulosa, consistent with most authors, until this re-evaluation is completed.All material we examined from four Governorates: Karbala, Basrah, Muthanna and Wasit correspond to C. tubulosa (in its current, most widely-accepted circumscription).None of the material we examined pertained to either C. salsa or C. flava, based on our careful consideration of the traits emphasized above.Similarly, none of the specimens tentatively labelled C. salsa corresponded with that species either (Fig. 2C); indeed, we only found evidence of glabrous plants, ruling out the occurrence of C. salsa in Iraq based on the material available.Furthermore, material from Iraq identified as C. flava in a doctoral thesis (Ataei 2017) appears to have been identified in error (Fig. 2A): the unbroken bracts do not greatly exceed the calyx; we believe detached bracts may have caused confusion; phylogenetic analysis later confirmed the specimen in question nested with C. tubulosa (Ataei et al. 2020).
Our extensive investigation based on herbarium specimens revealed that C. tubulosa occurs in every Governorate of Iraq except for the Kurdistan Autonomous Region.This could be due to the wide distribution of potential hosts across three of four main ecological regions, namely the deserts west of the Euphrates River, Upper Mesopotamia and Lower Mesopotamia (Ghazanfar and McDaniel 2016;Hegazy and Doust 2016).Cistanche has not been recorded in the fourth ecological region, the northern highlands of Iraqi Kurdistan.This region is an extension of the great Eurasian alpine system, and not a typical habitat for Cistanche which is primarily desert-dwelling; moreover, hosts typically associated with Cistanche -shrubby Amaranthaceae such as Haloxylon, are absent from this ecological region (Ghazanfar and McDaniel 2016;Hegazy and Doust 2016).We conclude from this examination that despite multiple reports of various taxa, only one species occurs in Iraq with certainty: Cistanche tubulosa.
It is of note that the corolla colour of C. tubulosa varies with age and population, from pale lemon yellow, to deep orange-yellow, with varying levels of pink to violet pigmentation.Similarly, the height and stature vary from 15 cm to 130 cm depending on rainfall and, potentially, host species.The key below is based on our observations of multiple populations across the region.

Figure 1 .
Figure 1.The locations of the material examined in Iraq.

Figure 2 .
Figure 2. A herbarium specimen of C. tubulosa collected in Iraq, mistakenly identified as C. flava B the holotype of putative species C. chabaharensis; note the woolly anther (inset) typical of C. tubulosa C herbarium specimen of C. tubulosa in (W) collected in lowland of Iraq, mistakenly identified as C. salsa.

Figure 3 .
Figure 3. Illustrations of Cistanche species putatively found in Iraq and adjacent territories (inflorescences) A C. flava B C. tubulosa C C. salsa D-F corollas in profile of C. flava, C. tubulosa and C. salsa, respectively; note calyx and bract characteristics.

Figure 4 .
Figure 4. Cistanche species putatively found in Iraq and adjacent territories A, B C. tubulosa (photographed in Iran) C, D C. flava (photographed in Kazakhstan) E, F C. tubulosa (photographed in Iraq; note excavated stem bases in E).Used with permission: photographs A, B by Sajad Alipour; photograph C by Lina Valdschmit; photograph D by Bobur Karimov.
(Ataei 2017) of Turkey(Davis 1982), C. salsa is the only species recorded.It is described as having densely lanate bracts and bracteoles, and a glabrous calyx and corolla.It was cited in three regions, two were in Inner Anatolia and one was in Kars city which is only 370 km from the Iraqi Kurdistan border.Therefore, C. salsa is a possible candidate species for the Flora of Iraq. 3. The Flora of Saudi Arabia (Migahid 1989) recorded two species, C. phelypaea (L.) Cout.(generally considered an Atlantic species) and C. tubulosa.Other species that are not included in the Flora have not been recorded for Saudi Arabia.These are C. violacea and C. rosea Baker (Foley 2004).The distribution of these species is narrow, C. rosea is distributed in the far south west and C. violacea in the northwest, far from the border with Iraq.Protologues for Cistanche eremodoxa, C. salsa, C. ridgewayana, C. fissa, C. laxiflora, C. flava, C. ambigua, C. lutea and C. phelypaea -the species identified as putatively in Iraq and adjacent regions of neighbouring countries -were examined as well the description of the unpublished putative species C. chabaharensis(Ataei 2017).Type specimens of these species were examined when they were available; type specimens of C. flava and C. tubulosa were unavailable, and are believed to be missing.The Natural History Museum, London (NHM) and the University of Vienna (WU) collections were examined, but they held no Iraqi specimens.An extensive survey of three herbaria: Kew (K), Edinburgh (E) and the Natural History Museum of Vienna (W) and examination of herbarium images from the National Herbarium in Baghdad (BAG) retrieved 62 specimens from Iraq.Specimens from adjacent countries were also examined for comparison.Images of living material in Iraq and adjacent countries sourced from social media were all examined, and cross-referenced with herbarium specimens.
(Daoud 1985)egion from the eastern Mediterranean seaboard to the Syrian Desert.This flora recorded C. lutea (Desf.)Hoffmanns.&Link,C.tubulosa and C. salsa.In the descriptions, C. lutea and C. tubulosa are described as glabrous and C. salsa as lanate.Of the three species, only C. lutea is reported to occur in the desertic regions contiguous with Iraq. 2. 4. The Flora of Kuwait(Daoud 1985)records only C. tubulosa, cites Soda rosmarinus (Bunge ex Boiss.)Akhani(listed under its synonym: Seidlitzia rosmarinus Bunge ex Boiss.(Amaranthaceae) as a host, and considers C. tubulosa var.tomentosaHook.f.i.c as a Synonym.Examination of protologues, types and representative specimens Moral et al. (2017) of Iraq and of adjacent countries, and of other relevant taxonomic studies, identified 10 published names and one unpublished name for species putatively in Iraq and adjacent countries.Species in adjacent countries, especially in Iran including, C. eremodoxa, C. ridgewayana, C. fissa, C. laxiflora and C. ambigua, were excluded from this investigation as their distributions and morphology suggest they are unlikely to exist in Iraq.Of the other species, two Atlantic/western European taxa were excluded on the basis that they are exceptionally unlikely to occur in the Middle East: C. lutea and C. phelypaea.Although C. lutea was cited in the Flora of Syria, Palestine and Sinai, the description does not provide adequate detail of morphological characters to discriminate between this and other species, including C. tubulosa.Cistanche phelypaea, as circumscribed currently, is a primarily Atlantic, coastal species, frequent from southwest Portugal south to Macaronesia and the coast of Morocco.We consider the inclusion of C. phelypaea in the Flora of Saudi Arabia to be equivocal, and cited with insufficient detail to merit further examination.MorenoMoral et al. (2017)consider C. lutea and C. phelypaea to be morphologically distinct.
Wood (1997)records the presence of C. phelypaea and C. rosea Bakir in Yemen and considered C. tubulosa a synonym of C. phelypaea.Author AM during his time in Yemen at the University of Taiz, in 2000-2002, observed a putatively distinct form of Cistanche across regions.It parasitized Halothamnus bottae Jaub.& Spach (syn.Salsola bottae (Jaub.& Spach) Boiss) (Amaranthaceae) (absent from Iraq) and was pure yellow, without purple pigmentation.Further investigation is required.Cistanche rosea, which is widespread across the Arabian Peninsula, is readily distinguished by its deep rose-red corolla.Ataei et al. (2020) asserts that C. lutea and C. phelypaea do not occur in the Middle East.Both entities were excluded from further work for the treatment of the genus Cistanche in Iraq.

Table 1 .
Key morphological characters of Cistanche species putatively found in Iraq and adjacent territories.