A new species from the W part of the Iranian Zagros Mountains in Ilam province, Dionysia robusta (Primulaceae), is described, illustrated and compared with similar and related species. It differs from these relatives in leaf shape, length and density of glandular hairs, and shape of the calyx. The DNA sequence of the nuclear ribosomal ITS region of D. robusta is most similar to that of D. gaubae.
Version of record first published online on 1 April 2016 ahead of inclusion in April 2016 issue.
Dionysia Fenzl, one of the larger genera of Primulaceae, consists of more than 50 species. They are suffrutescent herbs that form loose tufts or dense cushions in crevices of rocks and cliffs. During spring, these plants are covered by yellow, purple or pink flowers (Lidén 2007; Grey-Wilson 1989; Melchior 1943; Wendelbo 1961, 1964, 1965, 1971).
The majority of Dionysia species (more than 20) are found in the Zagros Mountains of W Iran (Lidén 2007). The number of species of Dionysia in other regions of SW Asia is lower: there are eight species in other areas of Iran, 11 species in Afghanistan, three in Turkey, two in Iraq, two in Turkmenistan, two in Tajikistan, one in Oman and one in Pakistan. The majority of species of Dionysia occurs in the area covered by Flora iranica (Wendelbo 1965). Thanks to the extensive collection of material from different mountainous areas of this region in recent years, new species have been described (Jamzad 1996, 1999; Lidén 2007; Borjian & al. 2014).
Ilam province in W Iran is a poorly studied area, from which several new species have recently been described (Mozaffarian 2008). Two nearby localities of an unknown Dionysia species were found in the Dinar-Kouh mountains in the Abdanan region of this province by the first author. Here, we report the finding of a new species and clarify its relationships to other species of Dionysia.
Material and methods
We compared our unknown species with other Dionysia species reported from W Iran and N Iraq, namely D. bornmuelleri (Pax) Clay, D. gaubae Bornm., D. odora Fenzl and D. tacamahaca Lidén (Wendelbo 1965; Jamzad 1999; Lidén 2007), as well as all Iranian species with similar morphology. All type specimens of the above-mentioned species were studied. Vegetative and floral parts were observed and measured under a stereomicroscope (NTB-3A, Wuzhou New Found Instrument Co., Guangxi, China). Voucher specimens were prepared and deposited in the herbarium of Science and Research Branch, Islamic Azad University (IAUH), Tehran, with duplicates in B and UPS (herbarium codes according to Thiers [continuously updated]).
Total DNA was extracted from either silica-gel dried leaves collected from the wild or leaf fragments of herbarium material using the NucleoSpin Plant II kit (Machery-Nagel, Düren, Germany) following the manufacturer's instructions. The complete internal transcribed spacer (ITS) region of the nuclear ribosomal cistron was amplified by using the forward primer AB101 (5′-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3′) and the reverse primer AB102 (5′-TAG AAT TCC CCG GTT CGC TCG CCG TTA C - 3′; Douzery & al. 1999) in a PCR reaction under the following conditions: a pretreatment of 5 min at 95 °C, 35 cycles of 30 sec at 95 °C, 30 sec at 50 °C, and 90 sec at 72 °C, and a final extension of 7 min at 72 °C. Amplicons were sequenced on an ABI 3730 DNA Analyzer (Hitachi-Applied Biosystems, Waltham, Massachusetts, U.S.A.). Sequences were visually checked and edited with the software tool Sequencher 4 (Gene Codes Corporation, Ann Arbor, Michigan, U.S.A.).
We added the ITS DNA sequence of the new species presented here to those of the species already present in the phylogeny of Dionysia by Trift & al. (2004). This new dataset was aligned using the software tool MacClade 4.08 (Maddison & Maddison 2000). A maximum parsimony (MP) analysis of the ITS dataset was performed via PAUP* (Swofford 2002) under the following parameters: heuristic search, number of replicates: 100, swapping method: TBR. The shortest trees recovered under MP were combined to form a strict consensus tree. Support for each branch was calculated via bootstrapping (Felsenstein 1985) using 100 replicates and the same setting as described above. Phylogenetic inferences were also conducted under the maximum likelihood (ML) criterion using the software tool RAxML 2.0 (Stamatakis 2014) on CIPRES Science Gateway (Miller & al. 2010). In addition, a Bayesian analysis was performed using the software tool MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003). Prior to phylogenetic analyses, the best-fitting model of DNA nucleotide substitution was selected using the software tool Modeltest 3.7 (Posada & Crandall 1998, 2001). Following this analysis, the following sequence model priors were set for the analysis in MrBayes: TrN+I+G (Nst = 6, rates=gamma, A–C substitution rate = 1.0000, A–G = 4.5110, A–T = 1.0000, C–G = 1.0000, C–T = 9.3590, G–T = 1.0000, proportion of invariable sites = 0.4147, shape parameter of the gamma distribution = 0.6374). A Markov Chain Monte Carlo (MCMC) sampling was conducted for 5 000 000 generations in four simultaneous chains, with trees being sampled every 100 generations for a total of 50 000 trees in the initial sample. The first 25 percent of the sampled trees were discarded, and the posterior probability of the resulting phylogeny and its branches was determined from the remaining trees.
Results and Discussion
Material of the new putative species showed some morphological dissimilarity to those species already described from W Iran, as summarized in Table 1. Morphologically, our material also showed resemblance to Dionysia iranica Jamzad and D. sarvestanica Jamzad & Grey-Wilson, which were described from localities further south. Genetically, the new material can be distinguished from these and other species, as shown in Table 2.
We found that the ITS DNA sequence of the new species is most similar to that of Dionysia gaubae and differs from it by only four bases (Table 2 and Fig. 1). MP analysis identified a set of 142 parsimony-informative characters and resulted in 449 most parsimonious trees with a length of 491 steps, a consistency index (CI) of 0.607 and a retention index (RI) of 0.749. The strict consensus tree displayed the following characteristics: length = 502 steps, CI = 0.594, and RI = 0.735 (tree not shown). In all three analyses of the ITS dataset (Fig. 1), our new putative species showed to be closely related to D. gaubae. It did not display close relationships to the morphologically similar species D. iranica and D. sarvestanica. Hence, we conclude that that our newly gathered material indeed represents a new species.
Dionysia robusta Younesi, sp. nov. — Fig. 2 & 3. Holotype: Iran, Ilam Province, SW of Abdanan, Dinar-Kouh protected region, Pizeleh, 32°56′09.6″N, 47°18′35.4″E, 1720 m, 11 May 2015, Younesi 14494 (IAUH; isotypes: B, TARI).
Description — Cushions very dense, bluish grey-green, up to 70 cm in diam. Branches tightly packed, very densely leafy, with cream-coloured marcescent leaves long persistent. Basal part of leaf suberect, distal part widely spreading, subspatulate-obovate, 4–7 mm long, 2–3 mm wide, covered with subsessile glands to 0.1 mm long, more sparsely so abaxially, margin entire (rarely slightly lobed at apex), apex obtuse to subacute. Inflorescence with a single sessile flower. Bract 1, oblanceolate, 4–5 mm long, c. 1 mm wide, base very shortly ciliate, margin entire or apically with a few minute denticles. Calyx 4–5 mm long, divided to ⅔-¾; lobes broadly lanceolate, often overlapping in basal ⅓, outside shortly glandular hairy, especially at base, on veins and on margin, inside sparsely glandular, base keeled, margin entire, apex subacute. Corolla yellow, glandular hairy, rather densely so toward base, less so apically, hairs 0.1–0.2 mm long; tube 20–24 mm long; limb c. 10 mm wide; lobes not or slightly overlapping, apex rounded or very slightly emarginate. Stamens in longstyled flowers inserted c. ⅔ from base; anthers c. 2 mm long. Ovary with 3 or 4 ovules; style in long-styled flowers conspicuously exserted; style in short-styled flowers c. ⅔ as long as corolla tube. Capsule broadly ovoid, c. 3 mm long, 3- or 4-seeded; valves thick, not twisted. Seeds 1.4-1.6 × 0.6-0.7 mm.
Phenology — Flowering in May, fruiting in June.
Distribution and ecology — Dionysia robusta is known from two localities in Ilam Province, Iran (Fig. 4). Each locality is a strip on the SE face of two parallel, deep gorges between 32°55′N, 47°18′E and 32°57′N, 47°22′E in an altitudinal range of 1550–1800 m. The type material was collected from the more northern strip. The species forms large subpopulations at both localities — not common for most species of this genus. The plants grow in crevices of vertical calcareous rocks and N-facing slopes. The general vegetation is dominated by Quercus brantii Lindl. accompanied by Acer monspessulanum L., Amygdalus orientalis Mill., Crataegus azarolus var. aronia L., Ficus carica sub sp. rupestris (Hausskn. ex Boiss.) Browicz and Pistacia atlantica Desf.
Comparison of morphological characters of Dionysia robusta, D. gaubae, D. iranica, D. sarvestanica and D. tacamahaca.
Comparison of different bases in ITS DNA sequences of Dionysia robusta, D. gaubae, D. iranica, D. odora and D. sarvestanica. — Alignment length = 681 bases; each column represents a position with dissimilar bases.
Conservation status — According to our observations, the number of mature individuals of Dionysia robusta is rather high (probably more than 10 000). We observed the population in two large localities in the form of two strips up to 10 km long and 400 m wide. The area of occupancy of D. robusta is less than 10 km2. There is no information on population size reduction, but we project a decline in the area of occupancy, quality of habitat, and number of mature individuals due to recent severe droughts in the area. We suggest, therefore, that this species should be placed under the IUCN (2012) category Critically Endangered with the criteria CR B2ab(ii,iii,v).
Etymology — The epithet robusta refers to the robust habit and large size of the plants.
Comparison with other species — Dionysia robusta is easily distinguishable from other species growing in the region by having cushions very large, up to 70 cm in diameter, and leaves entire or slightly divided. It is similar to D. gaubae, D. iranica, D. sarvestanica and D. tacamahaca (Table 1). However, D. gaubae differs in having leaves longer and usually more divided, marcescent leaves strongly reflexed so as to become more or less coiled, glands on the leaves more clearly stalked, and calyx much more deeply divided. Dionysia iranica differs in its dark green colour, usually less dense habit, and more deeply divided calyx. Dionysia sarvestanica differs in having leaves not or only slightly spreading, smaller, and always entire, more densely glandular with hairs clearly stalked, and calyx divided to the base. Dionysia robusta is similar to D. tacamahaca in the outcurved leaves and distinct calyx cup, but is very different in indumentum.
The authors would like to thank Libing Zhang (MO), an anonymous reviewer and Nicholas Turland (B) for their very accurate scrutiny of the earlier versions of this paper.
- Borjian A. , Deylami E. & Dousti A. F. 2014: Dionysia assadii sp. nov. (Primulaceae: sect. Dionysiopsis) from southern Iran. — Nordic J. Bot. 32: 717–722. Google Scholar
- Douzery E. J. P. , Pridgeon A. M. , Kores P. , Linder H. P. , Kurzweil H. & Chase M. W. 1999: Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences. — Amer. J. Bot. 86: 887–899. Google Scholar
- Felsenstein J. 1985: Phylogenies and the Comparative Method. — Amer. Naturalist 125: 1–15. Google Scholar
- Grey-Wilson C. 1989: The genus Dionysia. — Woking: Alpine Garden Society. Google Scholar
- IUCN 2012: IUCN Red List categories and criteria. Version 3.1, ed. 2. — Gland & Cambridge: IUCN. — Published at http://www.iucnredlist.org/documents/redlist_cats_crit_en.pdf Google Scholar
- Jamzad Z. 1996: The genus Dionysia (Primulaceae) in Iran. — Iran. J. Bot. 7: 15–30. Google Scholar
- Jamzad Z. 1999: Primulaceae — In: Assadi M. , Massoumi A. A. , Mozaffarian V. & Khatamsaz M. (ed.), Flora of Iran 25. Primulaceae. — Tehran: Research Institute of Forests and Rangelands. Google Scholar
- Lidén M. 2007: The genus Dionysia (Primulaceae), a synopsis and five new species. — Willdenowia 37: 37–61. Google Scholar
- Maddison D. R. & Maddison W. P. 2000: MacClade 4. — Sunderland: Sinauer Associates. — Published at http://macclade.org/ Google Scholar
- Melchior H. 1943: Entwicklungsgeschichte der Primulaceen: Gattung Dionysia. — Mitth. Thüring. Bot. Vereins 50: 156–174. Google Scholar
- Miller M. A. , Pfeiffer W. & Schwartz T. 2010: Creating the CIPRES science gateway for inference of large phylogenetic trees. — Pp. 45–52 in: 2010 Gateway Computing Environments Workshop (GCE 2010). New Orleans, Louisiana, USA, 14 November 2010. — Red Hook: IEEE (Institute of Electrical and Electronics Engineers). — doi http://dx.doi.org/10.1109/GCE.2010.5676129 Google Scholar
- Mozaffarian V. 2008: Flora of Ilam. — Tehran: Farhang Moaser. Google Scholar
- Posada D. & Crandall K. A. 1998: Modeltest: testing the model of DNA substitution. — Bioinformatics 14: 817–818. Google Scholar
- Posada D. & Crandall K. A. 2001: Selecting the best-fit model of nucleotide substitution. — Syst. Biol. 50: 580–601. Google Scholar
- Ronquist F. and Huelsenbeck J. P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. — Bioinformatics 19: 1572–1574. Google Scholar
- Stamatakis A. 2014: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. — Bioinformatics 30: 1312–1313. Google Scholar
- Swofford D. L. 2002: PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). — Sunderland: Sinauer Associates. — Published at http://paup.csit.fsu.edu/ Google Scholar
- Thiers B. [continuously updated]: Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. — Published at http://sweetgum.nybg.org/science/ih/ [accessed 1 Oct 2015]. Google Scholar
- Trift I. , Lidén M. & Anderberg A. A. 2004: Phylogeny and Biogeography of Dionysia. — Int. J. Plant Sci. 165: 845–860. Google Scholar
- Wendelbo P. 1961: Studies in Primulaceae I. A monograph of the genus Dionysia. — Aarbok Univ. Bergen, Mat.-Naturvitensk. Ser. 3. Google Scholar
- Wendelbo P. 1964: Studies in Primulaceae IV. The genus Dionysia in Afghanistan with descriptions of 6 new species. — Aarbok Univ. Bergen, Mat.-Naturvitensk. Ser. 19. Google Scholar
- Wendelbo P. 1965: Primulaceae — In: Rechinger K. H. (ed.), Flora iranica 9. — Graz: Akademische Druck- u. Verlagsanstalt. Google Scholar
- Wendelbo P. 1971: On xeromorphic adaptations in the genus Dionysia (Primulaceae). — Ann. Naturhist. Mus. Wien 75: 249–254. Google Scholar
Voucher information: GenBank accession numbers of material used in the molecular analysis of the ITS region. Except for two newly sequenced samples (indicated in boldface), all other accessions are from Trift & al. (2004) and Lidén (2007).
Dionysia archibaldii Wendelbo AY680737
D. aretioides (Lehm.) Boiss. AY680723
D. balsamea Wendelbo & Rech. f. AY680709 D. bryoides Boiss. AY680728
D. caespitosa (Duby) Boiss. AY680738
D. cristagalli Lidén AY680746
D. curviflora Bunge AY680739
D. denticulata Wendelbo AY680721
D. diapensiifolia Boiss. AY680713
D. esfandiarii Wendelbo AY680712
D. freitagii Wendelbo AY680729
D. gaubae Bornm. AY680740
D. haussknechtii Bornm. & Strauss AY680720
D. hissarica Lipsky AY680725
D. involucrata Zaprjag. AY680705 D. iranica Jamzad AY680741
D. iranshahrii Wendelbo AY680735
D. janthina Bornm. & C. Winkl. AY680711
D. khatamii Mozaff. AY680742
D. khuzistanica Jamzad AY680727
D. lamingtonii Stapf AY680743
D. leucotricha Bornm. AY680717
D. lindbergii Wendelbo AY680748
D. lurorum Wendelbo AY680718
D. michauxii (Duby) Boiss. AY680714
D. microphylla Wendelbo AY680706
D. mira Wendelbo AY680733
D. mozaffarianii Lidén AY680716
D. odora Fenzl AY680719
D. oreodoxa Bornm. AY680744
D. paradoxa Wendelbo AY680708
D. revoluta subsp. canescens (Boiss.) Wendelbo AY680730
D. revoluta Boiss. subsp. revoluta AY680731
D. rhaptodes Bunge AY680745
D. robusta Younesi1 KU697386
D. sarvestanica Jamzad & Grey-Wilson AY680715
D. teucrioides P. H. Davis & Wendelbo AY680734
D. viscidula Wendelbo AY680722
D. viva Lidén & Zetterl. AY680736
Primula davisii W. W. Sm. AY680710
P. floribunda Wall. AY680707
P. gaubaeana Bornm.2 KU697387
P. veris L. JQ927145