﻿Thliphthisasapphus (Rubiaceae, Rubieae), a new species from Lefkada (Ionian Islands, Greece) and its ecological position

﻿Abstract The new species, Thliphthisasapphussp. nov. (Rubiaceae, Rubieae), a narrow endemic of the white cliffs of Lefkátas on the southwest coast of Lefkada (Greece) is described and illustrated and an IUCN assessment is presented. Vegetation relevés were performed at the single known locality, limestone cliffs facing the sea and revealed a new association, the Thliphthisasapphus-Lomelosietumdallaportae. The chromosome number of Thliphthisasapphus was determined as 2n = 4x = 44, being the single tetraploid species in the genus to date. The species also differs markedly morphologically from its morphologically closest relatives, two Greek steno-endemic oreophytes, Th.baenitzii and Th.muscosa by the following characters: densely setose mericarps and corolla, tetraploidy and by its distribution. An identification key for the Greek species of Thliphthisa is provided. Th.sapphus constitutes the westernmost outpost of a group of Greek steno-endemics, highlighting the importance of coastal habitats and their protection as refugia for poorly competitive chamaephytes.


Introduction
The recently erected genus Thliphthisa (Griseb.)P.Caputo & Del Guacchio (Rubiaceae, Rubieae, Galiinae) is centred in the eastern Mediterranean and includes 22 species, mostly narrow endemics.The systematics and taxonomy of subtribe Galiinae with up to 900 species is complex and the genus boundaries are still disputed, but the group is generally recognised by herbaceous habit with pseudo-whorled leaves and dry mericarps (Ehrendorfer et al. 2018).According to traditional understanding, the group is divided into several small (e.g.Sherardia L.) to medium-sized genera (e.g.Asperula L.) and the large, para-and polyphyletic genus Galium L. with about 650 species.As a result of extensive morphological and phylogenetic studies, Ehrendorfer and co-workers recently proposed that several sections of Asperula and Galium be elevated to generic rank, which would allow the retention of all traditional genera of Galiinae such as Cruciata Mill., Sherardia or Valantia L., a viewpoint favoured over merging the whole Galiinae resulting in a large and heterogeneous Galium (s.latiss.)(Ehrendorfer and Barfuss 2014;Ehrendorfer et al. 2018).Following these suggestions, Galiinae should comprise more than a dozen small genera, but most formal taxonomic decisions following these results are still pending.
Asperula sects.Cynanchicae (DC.) Boiss., Thliphthisa (Griseb.)Ehrend.and Hexaphylla Klokov were consistently resolved as monophyletic groups together with the monophyletic Sherardia, but the relationships of these groups are not always resolved (Sherardia clade of Natali et al. (1995); Clade IV of Soza and Olmstead (2010); Clade VI of Ehrendorfer et al. (2018)).Following Ehrendorfer and co-workers, Del Guacchio and Caputo (2020) recently proposed to raise these sections to generic rank basically upgrading the "distinguishable smaller subclades" of Clade VI (Ehrendorfer et al. 2018) to generic level.Morphologically, Thliphthisa is variable and difficult to separate from other representatives of subtribe Galiinae, one of the few consistent characters being truncate fruits (Schönbeck-Temesy and Ehrendorfer 1985;Del Guacchio and Caputo 2020).In Thliphthisa, Del Guacchio and Caputo (2020) have combined all but one taxon treated in the synopsis (conspectus, key to species and distribution maps) of Schönbeck-Temesy and Ehrendorfer (1985), although without citing this revision.Only the African A. cyrenaica (E.A.Durand & Barratte) Pamp.was included in genus Hexaphylla (Klokov) P.Caputo & Del Guacchio.
In November 1994, during floristic and vegetation studies on the island of Lefkada, Ionian Islands, Greece, a presumably undescribed rubiaceous chamaephyte was found.At the time of discovery, the plants had passed flowering and only remnants of withered corollas and aborted fruits were found.Repeated study of the population in summer 1996 as well as spring 2000 and 2011, observations on plants grown in cultivation and herbarium studies comparing all other Mediterranean species of Asperula s.l.confirmed that the enigmatic taxon is a Thliphthisa species new to science.It is here described as Thliphthisa sapphus, its affinities, cytology and ecology are detailed and an identification key for the Greek species of the genus is provided.

Materials and methods
Due to the steep cliff terrain, direct observations are limited to a small part of the single known population of this new Thliphthisa species.Vegetation relevés using a modified Braun-Blanquet cover-abundance scale (see Barkman et al. (1964) for details) have been made at the habitats of the species in autumn 1994 and in spring 1996.These relevés have been combined into a table which was further compared to known vegetation types of chasmophytic vegetation described so far from the Mediterranean to establish the syntaxonomic community identity.
In 2011, a young plant from a secondary habitat in a road embankment above the cliffs was transferred to the Botanical Garden of the University of Vienna (thereafter HBV) for cultivation and karyological investigations.Chromosome numbers were determined applying the standard Feulgen staining technique as described in Weiss-Schneeweiss et al. (2009).Actively growing root tip meristems were pre-treated with a 0.002M aqueous solution of 8-hydroxyquinoline for 2.5 h at room temperature and 2.5 h at 4 °C, fixed in ethanol:acetic acid (3:1) for at least 3 h at room temperature and stored at -20 °C until use.Fixed root meristems were hydrolysed in 5N hydrochloric acid (HCl) for 30 min at room temperature, washed with tap water and stained with Schiff's reagent (Merck, Darmstadt, Germany) in darkness for 1 h (Jang et al. 2013).Chromosome spreads were prepared by squashing stained root tip meristems in a drop of 60% acetic acid under the cover-slip and analysed using an AxioImager M2 microscope (Carl Zeiss).All images were acquired with a CCD camera and files processed using AxioVision version 4.8 (Carl Zeiss).Karyotypes were made from these images in Corel Photo-Paint X5 (Corel Corp., Ottawa, Ontario).
For morphological comparison with the new species, specimens from the Herbaria W and WU of the other Thliphthisa taxa were examined with respect to diacritical characters as given in Schönbeck-Temesy and Ehrendorfer (1985).For a comprehensive list of analysed herbarium material from Greece, see Suppl.material 1.

Taxonomic treatment and discussion
Affinities of Thliphthisa sapphus.Due to the chamaephytic habit, with leaves (and leaf-like stipules) in rather closely-spaced whorls of 4-6, the presence of prophylls at the base of the sessile 4-merous flowers and the apically truncate ovary, Thliphthisa sapphus belongs to the subclade of the "Sherardia clade" (Manen et al. 1994;Natali et al. 1995), which is now classified as genus Thliphthisa (Del Guacchio and Caputo 2020).Within this group defined by Schönbeck-Temesy and Ehrendorfer (1985), the new species belongs to a group of chamaephytic taxa with presumably plesiomorphic flower structures, sharing well-developed Thliphthisa sapphus is most similar to Th. baenitzii, but is readily distinguished by mericarps and corolla being ± densely setose (vs.glabrous), by its longer corolla of approximately 3.5-4.5 mm (vs.2.5-3.5 mm in Th. baenitzii; Ehrendorfer and Krendl (1976), own measurements; the corolla length of 3-4 mm as given in Schönbeck-Temesy and Ehrendorfer (1985) is erroneous) and, in particular, by its tetraploid chromosome number that so far is unique within Thliphthisa (see below).Thliphthisa muscosa is also similar, but is well differentiated from both by its laxer growth, longer and ± erect flowering stems and distinctly longer, linear leaves.Distribution and biogeographic considerations.To our present knowledge Thliphthisa sapphus is restricted to a small area around the type locality on the western edge of the Lefkátas Peninsula in south-western Lefkada.Restricted distribution is common in the genus Thliphthisa which generally comprises eastern Mediterranean oreophytes distributed east of the Adriatic Sea and These three Hellenic steno-endemics form the north-western counterpart of the South Aegean Th. tournefortii and its local Cretan relative Th. crassula (the latter two constitute group A of Schönbeck-Temesy and Ehrendorfer (1985)).Thliphthisa tournefortii is a species of peculiar distribution (map in Runemark (1969: 115); Greuter (1971)) that can be associated chorologically with a "mesogaeic" element and, consequently, with a differentiation dating back to the Miocene (Greuter 1970).The same may be assumed for the above-mentioned more northern taxa Th. baenitzi, Th. muscosa and Th.sapphus sharing the more primitive flower structures with Th. tournefortii.The connections between these "sisters" are bridged geographically by another steno-endemic, still undescribed Thliphthisa species (Gutermann et al., in prep.) of Aetolia that, in habit and foliage, approaches the Cretan Th. crassula.Thus, Th. sapphus constitutes the westernmost outpost of this cluster of Greek steno-endemics.They underline the importance of cliffs as refugial habitats for poorly-competitive old chamaephytes (informally referred to as "Old ladies of the rocks") through the times (as e.g.discussed in Davis (1951) and Snogerup (1971)).
Etymology.The epithet commemorates Sappho, the most famous poetess of the Classical Hellenic Era and refers to the traditional (though non-historical) believe she suicidally threw herself from the white cliffs of Lefkátas because of unrequited love for beautiful young Phaon.The epithet is in genitive spelling.The genitive in classical Latin also is "Sapphūs", as testified in the poem delivered as "incerti avctoris epistvla Sapphvs ad Phaonem" in Ovid's Epistolae Heroidum no. 15.
Phenology and growth.Flowering of Thliphthisa sapphus commences in May and, by early July, mostly withering or wilted corollas were observed.At this time, mericarps were not developed even in wilted flowers, but some were conspicuously and unusually swollen and infected by parasites, possibly gall midges (Diptera, Cecidomyiidae) as found parasitising on other Rubiaceae taxa in the Mediterranean area (e.g.Simova-Tošić et al. (2000); Skuhravá and Skuhravý (2016); Skuhravá et al. 2020).The plant cultivated in the HBV never developed fertile fruits, thus suggesting self-infertility.However, we have also failed to find fully-developed fruits in situ.The propagation rate of Th. sapphus seems to be very low, as nearly all cushions observed within this area are old plants with a thick lignified rootstock and taproot (of 1 cm ∅ and more) anchored in the rock fissures (Fig. 2A).
Ecology and phytosociology.It dwells on steep to nearly vertical limestone cliffs facing the sea and is, thus, exposed to westerly winds, either in fissures in solid rock or within a thin regolith cover (Fig. 4).However, these habitats at elevations of 200-230 m a.s.l.border the slightly elevated plateau of the Peninsula and show no marked salt influence.Whereas large parts of the plateau are dominated by low Quercus coccifera L. garrigue, areas with shallower and rockier soils, especially along the upper slope of the coastal cliffs, are covered by sclerophyllous phrygana vegetation with Anthyllis hermanniae L., Erica multiflora L., Juniperus phoenicea subsp.turbinata (Guss.)Nyman or Thymbra capitata (L.) Cav.Depending on the distribution of narrow rock ledges, erosion fissures/ crevices and compact rock, the phrygana forms an intricate mosaic with sparse chasmophytic vegetation.As demonstrated by the vegetation relevés (Table 1), Thliphthisa sapphus is accompanied by another long-lived and cushion-forming chasmophyte Lomelosia crenata subsp.dallaportae (Boiss.)Greuter & Burdet showing scattered occurrences along the western coasts of the southern Ionian Islands (Flora Ionica Working Group 2016 onwards) and having also few isolated populations in Apulia (Italy) on Monte Gargano (Wagensommer 2012).Usually, together with Plocama calabrica (L.f.) M.Backlund & Thulin (Rubiaceae), they form a particular plant community covering the rock fissures.
Conservation status.In the area directly accessible to us, we counted about two dozen mature individuals of Thliphthisa sapphus.Within the range of sight, further cushions were identified in the nearly vertical cliff slopes below.The size of this single known population can be estimated at roughly 100 individuals, although more populations may exist in the inaccessible cliffs continuously extending north and south of the known population.In 2011, a few individuals Table 1.Thliphthisa sapphus-Lomelosietum dallaportae Mucina: vegetation relevés were performed using a modified Braun-Blanquet cover-abundance scale (see Barkman et al. (1964) for details) (10 November 1994: 1-4;16 May 1996: 5-11).

Other taxa
Lotus cytisoides L.

Mosses and lichens
were also found in Erica L. phrygana above the slopes and established on the gravelly banks along a close-by road above the primary cliff habitat.The occurrence on competition-free road banks suggests some potential of the species to colonise open anthropogenic sites, for which an occurrence in unstable gravelly habitats may have been a pre-adaptation.Due to the restricted known geographic range, the number of individuals may be drastically reduced by natural hazards, such as land or rock slides.The population is not exposed to immediate synanthropic threats, except for goats that graze even the steepest rock declivities such as the less precipitous slopes.Due to the small number of recorded individuals distributed within a range much less than 1 km 2 , the species falls into IUCN criterion D. Taking into account the possible threats mentioned above, it should be assessed as "Vulnerable" D2 (see IUCN (2022)).At national or regional scale, it classifies as a range-restricted endemic (as "r" in Dimopoulos et al. (2013)) and needs to be included in the "Red Data Lists" for Greece as "Endangered".Further monitoring of the population is certainly needed and conservation measures, such as reduction of grazing, should be taken for the subpopulation in the accessible area at the plateau above the ridge.

Figure 1 .
Figure 1.Illustration of Thliphthisa sapphus, based on the type material A whole plant B whole plant with vegetative and flowering shoots C vegetative shoot D detail of stem of vegetative shoot E flowering shoot F detail of stem of flowering shoot G flower H dissected flower (drawings by A. Kästner).

Figure 2 .
Figure 2. Habit and flowers of Thliphthisa sapphus A old individual of Th. sapphus with a thick lignified rootstock B flowers of Th. sapphus exhibiting the characteristic indumentum on the corolla (photographs by M. Sonnleitner).

Figure 4 .
Figure 4. Habitat and habit of Thliphthisa sapphus A coastal cliffs of the Lefkátas Peninsula, the locality of Th. sapphus (photograph by M. Hofbauer) B cushion of Th. sapphus in limestone rock fissure (photograph by M. Sonnleitner) C loose cushion of Th. sapphus within regolith cover on limestone rocks, together with Lomelosia crenata subsp.dallaportae (photograph by M. Sonnleitner).