Limonium artelariae (Plumbaginaceae), a new endemic species and further taxonomic and floristic notes on the genus in the island of Crete

Abstract: Some amendments of our knowledge of the taxonomically complex genus Limonium (Plumbaginaceae) in Crete, Greece are presented, based on field work and morphological study of herbarium specimens, including types. The circumscriptions of the closely related Cretan endemics L. cornarianum and L. hierapetrae are clarified. The former species is actually restricted to the type population in Moni Kapsa (SE Crete) and a newly found population in Pacheia Ammos (NE Crete). The latter species comprises populations in SE Crete that extend from Moni Kapsa to Dermatos, and also includes the populations previously assigned to L. chrisianum and L. minoicum that are found to be conspecific. Limonium artelariae from SE Crete, similar and related to L. cornarianum and L. hierapetrae, is described as a new species. It is rather unique among Greek Limonium species for its height (up to 110 cm tall) and its large, broadly spathulate leaves. Limonium cythereum previously known from the islands of Kythira and Antikythira is newly recorded for Crete, represented by two populations in NW Crete. The report of L. fragile from Crete is found to be erroneous, based on misidentified material of L. virgatum. All accepted taxa are described, and their types, chromosome number or ploidy level, distribution and ecology are also indicated. Photographs, distribution maps and an identification key are added to facilitate further identification. Citation: Koutroumpa K. 2024: Limonium artelariae (Plumbaginaceae), a new endemic species and further taxonomic and floristic notes on the genus in the island of Crete. – Willdenowia 54: 65–79 Version of record first published online on 13 March 2024 ahead of inclusion in April 2024 issue.


Introduction
Limonium Mill., comprising c. 600 species (Koutroumpa & al. 2018) is distributed worldwide (except Antarctica) and is by far the most species-rich and widespread genus in Plumbaginaceae.Its centre of diversity is the Mediterranean region, where c. 70% of the species occur, most of them local endemics of coastal habitats.The high species diversification of Limonium in the Mediterranean has been attributed to the combined effects of geoclimatic changes (i.e. the Messinian Salinity Crisis, the onset of the Mediterranean climate and the Plio-Pleistocene sea-level fluctuations) and its asexual reproduction via apomixis, according to recent macroevolutionary analyses (Koutroumpa & al. 2021).The occurrence of sexual and asexual reproduction and the ubiquity of hybridization and polyploidy in the Mediterranean species account for the notorious taxonomic complexity in Limonium (Pignatti 1971(Pignatti , 1972;;Erben 1979;Cowan 1998;Lledó & al. 2005Lledó & al. , 2011)).In such a genus, the clarification of species boundaries is often a very difficult task (Richards & al. 1996;Lledó & al. 2011).
Crete is the island with the highest number of Limo nium species in the entire E Mediterranean (see, e.g., Kouzali & al. 2012;Brullo & Erben 2016).It is home to 26 Limonium species, 11 of which are narrow endemics restricted to the Cretan coast and its offshore islets (Brullo & Erben 2016).As part of a systematics study of Limonium focusing on Crete and adjacent islands (S Aegean island arc), extensive field surveys (2014)(2015)(2016)(2017)(2018) were conducted, and the material evaluated.The resulting new insights into the taxonomy of the genus in Crete are provided here, including the description of a species new for science.

Material and methods
Fieldwork was conducted in Crete and adjacent islands (Chrysi, Gavdos, Karpathos, Kythira and Antikythira) during three visits from 2014 to 2018, and populations of Limonium species were recorded and collected.The field survey was particularly intensive on the island of Crete in order to cover the taxonomic and morphological variability of the genus.Herbarium specimens are deposited in the herbarium Z and duplicates in B (herbarium codes after Thiers 2023+).The current study is based on the investigation of the herbarium specimens from that fieldwork and other specimens in the herbaria B, C, FR, M, MSB and UPA, including type specimens.Fieldwork observations and detailed morphological studies of specimens informed species circumscriptions and taxonomic classifications, which were further backed up by the results of a phylogenomic study by Koutroumpa (2020).
Distribution maps were created using the R package "maps" (Becker & al. 2018;R Development Core Team 2019).Pollen and stigma type combinations of eight individuals of Limonium from two localities (Vatos and Kallikovrechtis) were examined in distilled water under an Olympus CX31 microscope (Olympus, Germany).Whether the population is monomorphic or dimorphic in terms of stigma-pollen floral types and whether the

Results and Discussion
The extensive fieldwork in Crete, combined with the morphological examination of several Limonium specimens and a comprehensive literature survey, highlighted some issues in the current classification of morphologically related Limonium species in SE Crete, and revealed the existence of a new undescribed endemic species.In addition, few species records for Limonium in Crete are updated.ferent highly supported clades (Koutroumpa 2020).The sample representing the coastal population is nested in the well-supported L. hierapetrae clade (Koutroumpa 2020).The confusion of the L. hierapetrae samples from Kapsa beach with L. cornarianum explains the significant differences between the species description given by Brullo & Erben (2016: 101) and the protologue of L. cornari anum by Kypriotakis & Artelari (1998: 144).Brullo & Erben (2016) did not see the holotype of L. cornarianum and assigned further populations growing along the coast from Ierapetra to Moni Kapsa to L. cornarianum (see, e.g., B [B 10 1189131]), which explains the much wider geographical distribution given by these authors for L. cornarianum that clearly overlaps with the known range of L. hierapetrae.In addition, the chromosome number 2n = 43 given for L. cornarianum (Brullo & Erben 2016) from material collected in Moni Kapsa matches the chromosome number of the pentaploid L. hierapetrae (2n = 43, Artelari 1989c), whereas L. cornarianum is triploid according to our initial studies (see below).The material used for the chromosome counts by Brullo & Erben (2016) was most probably collected from the coast and belongs to L. hierapetrae (see B [B 10 1132915]).Taken together, in the area of Moni Kapsa both L. cornarianum and L. hierapetrae co-occur (Fig. 1) but occupy rather distinct habitats: L. cornarianum is restricted to crevices

The circumscription and distribution of Limonium cornarianum
In the area of Moni Kapsa (Fig. 1), the narrow endemic Limonium cornarianum was originally described by Kypriotakis & Artelari (1998) from the calcareous rocks below the Kapsa monastery and the nearby gorge (named Moni Kapsa or Perivolakia gorge) that has an opening to the coast.Along the gorge, the species grows in crevices of steep cliffs (Fig. 2A) up to a distance of 1.5 km from the seashore (Kypriotakis & Artelari 1998).Later collectors (see e.g.specimens in Brullo & Erben 2016 and photos in Kypriotakis & Artelari 2009) have assigned also Limonium plants from the coastal habitats of Moni Kapsa to L. cornarianum.During our fieldwork, we observed that the individuals growing along the coast (Fig. 3) were morphologically different from the ones inside the gorge.The coastal populations fit the description of L. hierapetrae Rech.f. that has a wider distribution in SE Crete (Rechinger 1943;Artelari 1989c).These observations were verified by (1) morphologically examining and comparing specimens from Moni Kapsa and the surrounding areas, including the type material of L. cornari anum and L. hierapetrae, and (2) analysing samples from the Moni Kapsa gorge and the coast (Kapsa beach) in a phylogenomic study that revealed their placement in dif- of calcareous rocks (Fig. 2A) and L. hierapetrae grows on the nearby pebbly beach (Fig. 3) and in the maritime calcareous cliffs next to the beach.The two species can be easily distinguished by their different leaf shape (Fig. 4B,  C) and the smaller spikelets, spikelet bracts and calyces of L. cornarianum compared to L. hierapetrae (Table 1).
Until now, Limonium cornarianum was known only from its locus classicus (Moni Kapsa) in SE Crete, where it is rare, comprising only few individuals (Kypriotakis & Artelari 1998, 2009).The fieldwork in Crete revealed a new population of L. cornarianum in the NE part of the island (Fig. 1, 2B-D).The population is geographically and ecologically distinct from the one in Moni Kapsa and located at the E edge of Pacheia Ammos cove in a dry salt-marsh habitat (Fig. 1, 2B).Only few individuals (c.10) were found, occupying a small area that seems to be seasonally flooded and is the mouth of a stream that runs through the gorge of Cha, which is located about three kilometres from the seashore (Fig. 2B).Because L. cornarianum is a chasmophyte of steep, usually inaccessible cliffs free of other vegetation in its locus classicus, it could be assumed that the species might also occur inside the Cha gorge and that the population in Pacheia Ammos was formed by seeds washed down the stream that runs through the gorge.Cha is a narrow inaccessible gorge with high vertical cliffs and chasmophytic vegetation that is botanically not well explored because its passage requires canyoning expertise.The individuals of the newly found population in Pacheia Ammos undoubtfully represent L. cornarianum (Fig. 2C, D).They are mor-phologically indistinguishable and form a well-supported monophyletic group in the phylogeny with individuals from the type locality (Koutroumpa 2020).In addition, preliminary flow cytometric data of L. cornarianum from Pacheia Ammos indicate that the species is likely a triploid.Specifically, the 1C genome size of L. cornarianum is 2.37 pg, which is comparable to the genome size of the triploid L. virgatum (1C = 2.61 pg) and considerably smaller than the tetraploid L. aegaeum Erben & Brullo (1C = 3.15 pg) and the pentaploid L. sitiacum Rech.f. (1C = 3.52 pg).A triploid chromosome count is also found in L. aphroditae R. Artelari & Georgiou, an endemic in Kythira island that represents the closest morphological relative of L. cornarianum (Artelari & Georgiou 1999).Both species constitute a distinct morphological group compared to the other Aegean Limonium species, characterized by its small spikelet traits and sharing similarities with the C Mediterranean L. minutiflorum group that includes triploid and tetraploid taxa (Artelari & Georgiou 1999).The close relationship of L. cornarianum and L. aphroditae was further supported by phylogenomic data (Koutroumpa 2020).

The circumscription of Limonium hierapetrae
Limonium hierapetrae was described by Rechinger (1943) from a sandy beach next to Ierapetra in SE Crete.This endemic species was later found in a further five localities between Makrygialos and Nea Myrtos in the same wider area by Artelari (1989c), who studied the bio- systematics of the species, recorded its morphological variation and provided a detailed species description.Brullo & Erben (2016) described L. minoicum Erben & Brullo as a new species from SE Crete occurring between Tertsa and Tsoutsouros, a few kilometres W of the then known distribution of L. hierapetrae.According to the authors, the new species differs from L. hierapetrae by its shorter floral spikelets, inner bracts and calyces.However, Brullo & Erben (2016) gave a different description for L. hierapetrae than the one previously published by Artelari (1989c).Specifically, the measurements given for the sizes of leaves, spikes, spikelets, bracts and calyces by Brullo & Erben (2016) were only a subset of the size ranges given for L. hierapetrae by Artelari (1989c), namely the upper size ranges were assigned to L. hierapetrae and the lower size ranges were assigned to the newly described L. minoicum, for all the characters mentioned above.When Brullo & Erben's (2016) descriptions were employed to identify specimens collected along the coast between Moni Kapsa and Dermatos-Tsoutsouros (representing the entire range of L. hierapetrae and L. minoi cum), several of them belonging to previously identified L. hierapetrae populations were assigned to L. minoi cum or L. cornarianum sensu Brullo & Erben (2016), and many other specimens could not be unambiguously identified with either of them, but all perfectly match Artelari's (1989c) concept of L. hierapetrae.The phylogenomic analysis confirms that samples of L. hierapetrae and L. minoicum form one well-supported lineage, with L. minoicum specimens from the type locality and the population in Dermatos (bank of the river Anapodaris) intermingled with L. hierapetrae individuals from Moni Kapsa beach, Ferma and Ierapetra (i.e. the two species were not reciprocally monophyletic; Koutroumpa 2020).Additionally, chromosome counts of L. minoicum material with provenance from its locus classicus, M. Erben Li1708 (FR [FR-0128958], B [B 10 1009652]), share the same chromosome number (2n = 43) with L. hierape trae (Brullo & Erben 2016).Combining the morphological, molecular and chromosomal evidence, it becomes clear that L. minoicum is conspecific with L. hierapetrae.Also, L. chrisianum Brullo & Guarino, described from the islet of Chrysi S of Ierapetra, is conspecific with L. hierapetrae and represents just a small form of it, because the morphological features previously used to dif-ferentiate it from L. hierapetrae (e.g.smaller size and leaf length, dense arrangement of spikelets and larger middle bract width, Brullo & Guarino 2000) clearly fall within the range of variation of the latter species.

A new endemic Limonium species in SE Crete
During the fieldwork in summer of 2014, an impressive population of Limonium was found in SE Crete, in the area between Myrtos and Tertsa, comprising tall plants up to 110 cm, bearing large, broadly spathulate rosette leaves (Fig. 5).Resembling L. hierapetrae in terms of habit and inflorescence, its leaves are of a different shape, resembling more those of L. cornarianum (Fig. 4A, B).However, plants of such height and with leaves of such size found in that population are unique among the Li monium species in Greece belonging to the "Mediterranean lineage", i.e. all Greek Limonium species apart from eight belonging to the morphologically and phylogenetically distinct L. sect.Limonium (L.vulgare group), L. sect.Nephrophyllum Rech.f. s.l. and L. sect.Pterocladus (Spach) Bokhari (Koutroumpa & al. 2018).Three representatives sampled in a phylogenomic study formed a well-supported monophyletic group in close evolution- ary relationship with L. cornarianum and L. hierapetrae but clearly distinct from them (Koutroumpa 2020).Apart from the large population found during the fieldwork, a careful study of previously collected herbarium specimens revealed that material collected from Cape Theophilos (Brullo & Giusso s.n., herb. Erben) at the E edge of Sidonia beach, which is located to the W of Tertsa, also belong to the new species.In addition, plants grown from seeds of the Cape Theophilos collection were studied karyologically by M. Erben (Li-1721) who counted the pentaploid chromosome number 2n = 43 in a cultivated individual (voucher deposited in FR [FR-0128957]).The pentaploid chromosome number of the new species agrees with the preliminary flow cytometric analysis of material collected from Vatos and Kallikovrechtis, two localities between Myrtos and Tertsa.Specifically, the 1C genome size of the new species is 3.67-3.69pg, which is comparable to the genome size of the pentaploid (2n = 43) L. sitiacum (1C = 3.52 pg) but larger than the genome sizes of the tetraploid (2n = 34) L. aegaeum (1C = 3.15 pg) and the triploid (2n = 27) L. virgatum (1C = 2.61 pg) and smaller than the genome size of the hexaploid L. creticum (1C = c. 4 pg; this an approximate count because this genome size was partially overlapping with the Pisum sativum reference genome, 1C = 4.42 pg).In the Taxonomic treatment below, the new species is described and compared with the closely related L. cornarianum and L. hierapetrae, for which emended species descriptions are provided based on the plethora of material seen and examined, including the type specimens.

Limonium cythereum newly recorded for Crete
The endemic Limonium cythereum R. Artelari & Georgiou was until now known only from the islands of Kythira and Antikythira (Artelari & Georgiou 1999;Brullo & Erben 2016), which are located SE of Peloponnese and between Kythira and NW Crete, respectively (Fig. 6).Apart from the three known populations of L.

Limonium fragile was erroneously reported for Crete
Limonium fragile Erben & Brullo is a small species, up to 15 cm tall, bearing delicate branches, and has been reported from only few localities in Karpathos, Crete and Antikythira islands by Brullo & Erben (2016).Its occurrence in Crete is based on a specimen collected from the saline habitats of Olous near Elounda in NE Crete (Hansen 1066, PAL-Gr!; Brullo & Erben 2016).The specimen was originally identified as L. virgatum by W. Greuter in 1974, but Brullo & Erben (2016) later assigned it to L. fragile possibly on the basis of its small size.Hybrids between L. fragile and L. virgatum have also been reported from Crete by Brullo & Erben (2016), based on material collected from the same locality (i.e.salt marshes in Olous, H. Kalheber 99192, herb.Kalheber!).The latter specimen comprises immature individuals collected in early spring.Olous and the surrounding areas in Elounda were visited twice during the fieldwork, but an extensive search failed to find representatives of L. fragile.However, a very large population of L. virga tum was observed with plants of different sizes, some of them very small, growing on the saline basins that were seasonally flooded.All the material collected from this region clearly represents L. virgatum.Field observations and the examination of several specimens from Elounda, including the Cretan L. fragile specimens seen by Brullo & Erben (2016), support the view that small and immature individuals of L. virgatum from Olous were misidentified as L. fragile or L. fragile × L. virgatum by Brullo & Erben (2016).Therefore, L. fragile is to be regarded as absent from Crete and plants growing in Olous (Elounda) in NE Crete belong to L. virgatum.Diagnosis Limonium artelariae is distinguished from its closely related species in SE Crete, L. cornarianum and L. hierapetrae, by its broadly spathulate, rounded leaves and broadly obovate and wider inner spikelet bracts.Limonium artelariae is rather unique among all other Aegean Limonium endemics for its predominantly tall habit combined with longer and wider, spathulate leaves.
Breeding system -Apomixis is inferred from the monomorphic population with self-incompatible pollen-stigma combination B. In addition, the species is of high ploidy level with an odd chromosome number further supporting the view of an apomictic reproduction, following Erben (1978Erben ( , 1979)).
Distribution -Endemic to SE Crete, along the coast between the villages of Myrtos and Tertsa (replaced by Limonium hierapetrae in Tertsa), and at Cape Theophilos at the E edge of Sidonia beach (Fig. 1).
Habitat -Maritime cliffs of marly, calcareous, conglomerate and sandy substrates.
Eponymy -The specific epithet "artelariae" honours Dr. Rea Artelari, a former Associate Professor of the University of Patras, Greece, for her invaluable contributions to the systematics of Limonium in Greece and Cyprus throughout her academic career.
Taxonomic notes -The new species is morphologically most closely related to Limonium hierapetrae, which grows in the same area of SE Crete.However, L. artelariae has large, broadly spathulate, rounded leaves compared to the smaller, oblanceolate to oblong-obovate, mostly obtuse leaves of L. hierapetrae.The individuals of the latter are also smaller and have usually narrower, elliptic to oblong-obovate inner spikelet bracts (vs broadly obovate in L. artelariae; Table 1).The dense leaf rosettes with spathulate, sometimes irregularly folded leaves, the densely arranged inflorescences and the obovate shape of the inner spikelet bracts morphologically link L. arte lariae to L. cornarianum, which also grows in SE Crete.Further, L. cornarianum is clearly distinguished from L. artelariae by its smaller spikelets and spikelet bracts, and leaves with an acute apex (Table 1, Fig. 4).

Limonium hierapetrae
Chromosome number and ploidy -Ploidy level: 3x (inferred from preliminary flow cytometric results of genome size).
Breeding system -Apomixis was inferred from the monomorphic population in the locus classicus with selfincompatible floral combination B (Artelari & Georgiou 1999) and triploidy.
Habitat -Steep, calcareous rocks and dried salt marsh near the coast.
Additional specimens seen -Greece, Crete, prefecture of Lasithi: Pacheia Ammos, at E side of coast, on salt marshes between little church Agia Foteini and a small port, marshes with stigma-pollen combinations are self-incompatible (combinations A and B sensu Erben 1978) or self-compatible (combinations C and D sensu Erben 1978) could indicate the reproductive system (i.e.sexual [outcrossing or selfing] or asexual [apomictic]) of Limonium in the two aforementioned localities.In addition, a preliminary flow cytometry analysis (protocol by Temsch & al. 2010 with minor adjustments) was performed to estimate the genome sizes of L. cornarianum Kypr.& R. Artelari and the morphologically distinct Limonium in Vatos and Kallikovrechtis and compare them to the genome-size estimates of the triploid L. virgatum (Willd.)Fourr.(2n = 3x = 27; Artelari & Georgiou 2003), the tetraploid L. aegaeum Erben & Brullo (2n = 4x = 34; Brullo & Erben 2016), the pentaploid L. sitiacum Rech.f. (2n = 5x = 43; Brullo & Erben 2016) and the hexaploid L. creticum R. Artelari (2n = 6x = 61; Artelari 1989b), aiming at inferring their ploidy level.One plant per species collected from the wild and grown at the greenhouse of the Botanical Garden of Zurich was measured, except for the morphologically distinct Limonium in Vatos and Kalli kovrechtis for which two plants grown from seeds of wild origin, one plant from each locality, were measured.Pisum sati vum "Kleine Rheinländerin" (1C = 4.42 pg; Greilhuber & Ebert 1994) was used as reference, and all measurements were done in a Cyflow Space (Sysmex-Partec) flow cytometer.

Fig. 2 .
Fig. 2. Limonium cornarianum -A: individual growing on steep calcareous cliffs at the gorge of Moni Kapsa (locus classicus); B: habitat in Pacheia Ammos (new locality) with the Cha gorge in the background; C, D: inflorescence and habit of an individual in Pacheia Ammos.-All photographs taken by and © Konstantina Koutroumpa.
cythereum on the S and E coasts of Kythira (Chalkos, Vrouleas, and Avlemon) and the single population on the N coast of Antikythira (Potamos), the fieldwork revealed the occurrence of L. cythereum in two localities in NW Crete (briefly mentioned in Koutroumpa 2020).The species was recorded from a small port at the E edge of the city of Chania (see, e.g., B [B 10 1163863]) and Kalathas beach, which is few kilometres NE of Chania (see, e.g., B [B 10 1163868]).Limonium cythereum is easily distinguished from the closely related L. sieberi Kuntze, which also occurs in NW Crete and co-occurs with L. cythereum at Kalathas beach, by its shorter spikelets, the hairy and shorter inner spikelet bracts and the shorter calyces.The current distribution of L. cythereum reflects the phytogeographical connections of Crete and Kythira and the paleogeography of the region during the Pleistocene (c.0.8 Mya) when low sea levels during the glacial period reconnected islands with each other and some islands with the mainland (see maps inValakos 2008).