Two new infraspecific taxa of Verbascum delphicum (Scrophulariaceae, Scrophularieae) from mainland Greece and the island of Evvia

Abstract Verbascum delphicum Boiss. & Heldr. subsp. cervi Zografidis (Scrophulariaceae, Scrophularieae) is described as a subspecies new to science and illustrated. It is narrowly distributed in the Greek National Park of Mt Parnitha (Attica, Greece) with a very small population size. The new subspecies is a seldom-collected taxon, previously overlooked and misidentified as consubspecific with the autonymous subspecies, an endemic of the island of Evvia (Greece). Also described in this study is a new variety of subsp. delphicum from Mt Ochi of southern Evvia.


Introduction
Comprising more than 360 species, Verbascum L. (Scrophulariaceae) is the largest genus of the predominantly northern temperate tribe Scrophularieae Dumort. (= Verbasceae Dumort.) and is chiefl y represented by rosulate, biennial or perennial herbs with yellow-fl owered, thyrsic or racemose infl orescences (Fischer 2004;Oxelman et al. 2005). Although the genus is widely distributed in Eurasia and North Africa, species diversity is mostly concentrated in a region encompassing Anatolia, Caucasus, northwestern Iran, the Levant and the southern Balkans (Murbeck 1939). In Greece, Verbascum is among the 10 most species-rich and the 10 most range-restricted-rich genera with a total of 76 species and an endemism rate of 32% (Dimopoulos et al. 2013).
Verbascum delphicum Boiss. & Heldr. was discovered by Th eodor von Heldreich on Mt Dirfi of the island of Evvia (West Aegean Islands, Greece) and was subsequently jointly described by himself and Edmond Boissier in Boissier's Diagnoses plantarum Orientalium novarum (Boissier 1856). Th e autonymous subspecies is a nemoral, shortlived perennial or biennial herb with often impressive, sizeable basal leaves. Knowing the taxon well from my excursions on the mountains of central and south Evvia I was surprised to fi nd on Mt Parnitha (Attica, Sterea Ellas, mainland Greece) on May 2013 a small population of a closely related, yet distinct and undescribed as it appeared to be taxon. Th e population consisted of biennial plants with noticeably smaller and more or less diff erently shaped leaves than those of the insular V. delphicum. Inspection of the mainland population in the past recent years and comparison with the insular populations revealed some additional deviating traits for the taxon on Mt Parnitha. Taking into consideration overall morphology and the common habitat and phenology of the two taxa, as well as the phytogeographic connection between Sterea Ellas and Ins. Evvia, I propose that collectively the diff erences indicate distinction at the subspecies rank.
Th e new subspecies V. delphicum subsp. cervi Zografi dis described and illustrated in this work was apparently overlooked and misidentifi ed by Murbeck as consubspecifi c with subsp. delphicum based on material collected by Heldreich and Bassilios Tuntas from Mt Parnitha. Th e diff erences between the two subspecies are presented and discussed. A new, distinct variety of subsp. delphicum from Mt Ochi, south Evvia is also described.

Methods
For the cultivated material current-year seeds were sown in early October (2013 & 2015), in small pots fi lled with Compo® Bio Anzucht-und Kräutererde substrate. Twoweeks old plants were individually transferred in plastic pots 7 cm of diameter and were transplanted every c. 8 weeks two additional times in successively larger plastic pots, 15 and 24 cm of diameter. Th e plants were kept outdoors, daily receiving 5 hours of direct sunlight on average, whereas the substrate was kept moist but not soggy.
Measurements were performed either with a common ruler or under a stereomicroscope (Zeiss, Stemi 2000-C) equipped with a camera and ImagePro software. Because the population size of the new subspecies is extremely small the measurements were either performed in situ without damaging the plants, or, when this was not feasible, only small plant-parts were collected for subsequent assessment under the stereomicroscope. In particular, two fl ower-clusters (glomerules) of the middle part of the infl orescence were removed from 20 individual plants i) at fl owering and ii) at fruiting. Statistical signifi cance was assessed with two-tailed T-tests for two independent means (p<0.01) and two-tailed Mann-Whitney U-tests (p<0.01). Diagnosis. subsp. cervi can be distinguished from subsp. dephicum by a combination of the following characters: lamina of larger rosette-leaves 6-22 × 3-11 cm (vs. 16-40 × 11-24), 1.5-3.7 of length to width ratio (vs. 1.2-1.9), obtuse-cuneate at the base (vs. obtuse-truncate to cordate); indumentum of abaxial surface of rosette-leaves thinner; indumentum of adaxial surface of fi rst-year mature rosette-leaves ± harsh (vs. soft); stamen fi laments greenish-white (vs. orange).
Etymology. Name derives from the genitive of the Latin word "cervus" in reference to the red deer of the National Park which were often observed in the subspecies habitat. Th e popular animals are a considerable threat to their own Verbascum as they consume the young infl orescences. Description. Polycarpic eglandular-and minutely glandular-hairy short-lived perennial herb -or less often monocarpic biennial-producing a well-branched taproot and a sterile, erect or ascending, terete, leafy stem in the fi rst year of vegetative growth, followed by the production of an additional sterile stem and a terete fl owering-stem in each of the succeeding few years of vegetative and reproductive growth. Eglandular hairs dendritic, 0.4-2 mm of length, more or less covering the whole aerial part of the plant; glandular hairs minute, sparse, present on leaves, bracts, bracteols and calyx segments, visible by microscopy; fully developed fi rst-season leaves whitish-to yellowish-and softly-tomentose above, grayish-white and softly tomentose beneath; sterile stems up to 80 cm in height; fertile stems 40-180 cm of height, green to reddishblack, glabrescent but ± persistently, tomentose below; lower cauline-leaves petiolate; petiole up to 15 cm of length; lamina ovate to widely ovate, 1.2-1.9 of length to width ratio, obtuse-truncate to cordate at the base, crenate, obtuse at apex; larger leaf laminas 16-40 × 11-24 cm; middle cauline-leaves similar but progressively smaller and often subacute at the apex, shortly petiolate; upper cauline-leaves small, sessile, ovate-cordate, subacute at the apex; all cauline leaves alternate; Infl orescence 25-60 cm of height, simple or sparingly branched at the base with short, sub-erect branches and then infl orescence narrowly pyramidal in outline; fl owers arranged in clusters of pedicellate, compacted cymes (glomerules), ± crowded at least above, consisting of 3 -12 fl owers; bracts 3-7 × 2-4 mm, ovate to lanceolate, acute to acuminate or cuspidate, glabrescent; bracteoles present, similar to bracts but smaller; longer pedicels 4-10 mm of length, tomentose, ± glabrescent; calyx divided almost to the base into 5 lanceolate to lanceolate-linear, acute segments, 3.5-6 × 1.1-1.7 mm; abaxial surface of calyx tomentose, ± glabrescent; corolla rotate, fl at, 1.5-2.8 cm of diameter, yellow, with or without indistinct purple marks on the throat, with pellucid glands, divided to c. 3/4 into 5 broadly-obovate, subequal lobes; abaxial surface of corolla partially tomentose, adaxial surface sometimes ciliate near the throat, otherwise glabrous; tube of corolla ± infundibuliform, 1-2.3 mm of length, 1.5-2.2 mm of diameter; stamens 5, free, ciliate with white, clavate hairs which reach the connective of all anthers or do not reach the connective of the anterior stamens; three posterior stamens 5-8 mm, two anterior stamens 6-10 mm; stamen fi laments orange; all fi ve anthers reniform, mediofi x, papillose on adaxial surface, or glabrous on adaxial surface of the connective of anterior stamens, 0.8-1.4 mm; style tomentose at the base, 6-9 mm, slightly clavate at the apex; stigma hemispherical; capsule (excl. rostrum) 3.5-7 × 3-5 mm, ovoid to broadly ovoid, densely tomentose on early development, later glabrescent, with a rostrum 1-1.5 mm; seeds numerous, chestnut brown to dark brown, 0.7-1.1 × 0.5-0.7 mm, obpyramidal to ovoid-oblong, irregularly prismatic, faveolate with 4-8 pits in each longitudinal series.

Verbascum delphicum
Etymology. Name is in reference to the often abounding in ferns habitat of the variety.

Results and discussion
Th e diff erences between V. delphicum subsp. cervi and subsp. delphicum are presented in Table 1, whereas a comparative illustration is presented in Figure 2. A three-year fi eld survey of both insular and mainland populations and examination of cultivated plants confi rmed the constancy of the distinctive features. Subspecies cervi is characterized by its smaller and more elongated leaves with the base of leaf-laminas obtuse to long-cuneate -vs. ± truncate or even cordate in subsp. delphicum-and by a shorter, harsher and darker indumentum. Another signifi cant character is the color of the stamen fi laments: either greenish-white in subsp. cervi or deep-orange in subsp. delphicum. I note that this character is almost lost with drying. Life-cycle is an additional interesting trait. In his Flora Orientalis, Boissier has cited V. delphicum as a biennial herb (Boissier 1856), a vague conception which has been perpetuated in various fl oras ever since (Ferguson 1972;Raus 1986). Although biennial individuals are indeed found among its populations, the autonymous subspecies is more frequently a short-lived perennial herb as evidenced by the concomitant existence of the fl owering stem and either of an additional leaf-rosette or of a previous-year withered infructenscence or of both on the same plants. On the other hand, subsp. cervi has clearly drifted towards the biennial life-cycle and very rarely do the plants exceed the usual two years lifespan -I have seen only two such individuals.
On Mt Parnitha and on the mountainous region of central Evvia -Mts Dirfi and Xirovouni-V. delphicum inhabits Greek-fi r or mixed Abies-Castanea woodlands, fl owers from early-mid-June to early-mid-August and matures seeds from July to August. On Mt Ochi of southern Evvia the species is found in open Castanea woodlands -Abies is absent from southern Evvia-or in sub-montate to montane, open shrublands dominated by thick patches of Pteridium aquilinum ferns. In these habitats of Mt Ochi, I have found that subsp. delphicum frequently produce sterile stems instead of basal leaf rosettes; the unusual trait gives a quite distinctive appearance to the plants but apart from it they are indistinguishable from typical individuals that grow in proximity. I propose this taxon to be classifi ed as a new variety of subsp. delphicum ( Figure  3 and Supplementary fi le 2). Th e variety might have evolved from the competition for sunlight inside the dense patches of ferns of these habitats, whereas I have never found it in central Evvia -not even in the shadier spots where subsp. delphicum produces the typical basal rosettes. Murbeck's assumption that typical V. delphicum existed on Mt Parnitha was based on the following material he cited in his monograph: i) a collection by Heldreich -"Parnes, reg. abiet. occid., leg. HELDREICH 1854, n. 2902"-even though Heldreich himself never reported V. delphicum from mainland Greece and the date of the collection, which has unfortunately been destroyed during World War II (Th . Raus, personal communication, 27 August 2015), precedes by two years that of the original description of the species; ii) three collections by collector Bassilios Tuntas -" Parnes, reg. abiet., occid., leg. TUNTAS 5/7/1911, n. 1247Hayek].-Mt Parnes, prope cacumen, leg. TUNTAS 21/7/1907, n. 356 [Hb. Hal.];13/6/1909, n. 810 [Hb. Hal.]"-which, however, were not found either in WU-Generale, WU-Halácsy-Graecum or WU-Halácsy-Europaeum (W. Till, personal communication, 15 March 2016). Perhaps the only surviving voucher is kept in the Gothenburg Herbarium and likely corresponds to the aforementioned n. 1247 specimen of the collector, although the serial number is missing. Nevertheless, on the sheet there is a label with Murbeck's name and handwriting that states "V. delphicum". Having seen the specimen I have no doubt it represents subsp. cervi on account of the characteristic small basal leaves with an obtuse-cuneate base and the short indumentum. It is interesting to note that the aforementioned specimen includes the whole aerial part of a plant arranged on a single sheet, which is actually feasible for a medium individual of subsp. cervi -e.g., see the Holotype, Supplementary fi le 1. On the contrary, a medium individual of subsp. delphicum is impossible to completely fi t on a voucher sheet because the basal leaves are too big. I conclude that on Mt Parnitha there exists a single infraspecifi c taxon of V. delphicum, namely subsp. cervi, allopatric to the insular subsp. delphicum which is confi ned to Evvia. Th e species had not been collected from the mainland for almost a century, but its existence was confi rmed by a recent fl oristic study on the mountain (Aplada et al. 2007), (E. Aplada, personal communication 1 September 2016). Other reports of V. delphicum from the mainland -from the Peloponnese cited in (Dimopoulos et al. 2013) or from Mt Pendeli cited in (Vladimirov et al. 2012)-are in error (Th . Raus, and K. Polymenakos, personal communications respectively). Th e distribution map of V. delphicum is presented in Figure 4.

Suggested conservational status
V. delphicum subsp. cervi is restricted to the core of the National Park of Mt Parnitha where it apparently constitutes a single, fragmented population within an area of occupancy of less than 1 km 2 and a total population size of no more than 50 mature individuals (August 2016). Th e alarmingly rare new taxon has suff ered habitat loss with the devastating fi re of 2007 on the mountain and is now under direct threat by the overpopulated deer of the National Park which consume the developing infl orescences; the bitten plants respond by producing a few new small fl owering shoots but seed yield is expected to be severely aff ected. Importantly, the population size of V. delphicum subsp. cervi has been reduced about 20% over the last three years and the taxon is apparently critically endangered according to IUCN Red list criteria C(2a(i,ii)) and D(1) (IUCN 2001).

Comments on taxonomic affinities and phytogeography
Considering the recent radiation in the genus Verbascum (Ghahremaninejad et al. 2014) and the strong phytogeographic connection between Ins. Evvia and Sterea Ellas (Trigas 2003), it is reasonable to assume that V. delphicum subsp. cervi represents a neoendemic taxon that has diverged from the insular subspecies through the process of allopatric subspeciation. On the other hand, the infrageneric relationships of V. delphicum are more diffi cult to infer. In particular, Murbeck places V. delphicum under Sect. Bothrosperma Murb., Subsect. Fasciculata Murb., B. Isandra Franch., p. p., a. Bracteolata Murb., α Umbellulifera Murb., 1. Adenanthera Murb. and further groups it with the widespread in the Balkans V. banaticum Schrad. on account of the white stamen-hairs and the relatively long pedicels. However, the two species are very different and only doubtfully closely related -V. banaticum is an eglandular herb with lobed and pinnatisect at the base of the lamina rosette-leaves, freely branched, leafy, lax infl orescences and has smaller all fl ower parts and capsule. Rather I suspect that Adenanthera -i.e. having papillate connective of the anthers of the anterior stamensis a non-monophyletic trait and that V. delphicum is actually more closely related to some Leianthera Murb. -i.e. with glabrous connective-species of the centered but not restricted to the Balkans group of V. nigrum L., V. chaixii Vill., V. lanatum Schrad. and V. glabratrum Friv. Th ese species show considerable resemblance to V. delphicum with respect to general leaf morphology, indumentum and habit. Moreover, additionally to their glabrous connective of anterior stamens, these species all diff er from V. delphicum in the purple stamen-hairs and in the more or less striate stems. I should also note at this point that the connective of the anterior stamens of V. delphicum is often glabrous too and this is particularly evident for subsp. delphicum - Figure  2F shows one such stamen with the hairs not found on the connective. Intriguingly, V. delphicum also shows considerable phenetic resemblance with two Asiatic species, namely V. discolor Murb. from the Amanus Mountains and V. antitauricum Hub.-Mor. from the Anti-Taurus mountains, both of south-central Turkey. V. discolor is generally a more glabrous plant than V. delphicum with glabrous connective of anterior stamens, glabrous outer surface of the corolla and a more branched and laxer infl orescence. V. antitauricum, similarly to V. delphicum has either papillate or glabrous connective of anterior stamens but has bicolored stamen-hairs, glabrous corollas on the outer surface, more or less shorter pedicels and it is eglandular. In any case, the diff erences between V. delphicum and the Turkish species are not more pronounced than those between V. delphicum and the aforementioned Balkan species -compared individually-and thus additional data from molecular phylogenetics and/or cytological studies are needed for clarifying the topic of V. delphicum infrageneric affi nities.

Conclusions
Both infraspecifi c taxa of V. delphicum described in this study are distinct and easily recognized, whereas the species closest relative remains unknown. Subspecies cervi is apparently a critically endangered taxon.