An updated checklist of the lichens of St. Eustatius, Netherlands Antilles

Abstract In the course of a multi-taxon biodiversity inventory for the island of St. Eustatius, lichens were collected from 11 plots representing different vegetation types. From these collections, 126 lichen species are reported, 54 of which are new reports for St. Eustatius. Most species could be identified to species level based on morphological and chemical characters. In a few cases, mtSSU DNA sequences were generated for a preliminary molecular identification and future phylogenetic studies. In total, 263 identified lichen species are currently known from St. Eustatius, as well as some additional genera with yet unidentified species and lichenicolous fungi.


Introduction
Sint Eustatius is a small island (21 km 2 ) in the northern Leeward Islands part of the West Indies. It is one of the six islands of the Netherlands Antilles and, since 2010, a special municipality of the Netherlands. Sint Eustatius is roughly divided into three parts, the Northern Hills, the urbanised central area ('Cultuurvlakte') and the southern part dominated by the steep dormant volcano The Quill (600 m elev.). Although the whole island of St. Eustatius has been heavily impacted by human activities, the northern and M7−M9) on and around The Quill in the southern part of the island and one (U1) in the central urban area. Details concerning the location and vegetation characteristics of the plot locations and the multi-taxon sampling approach are described in van Andel et al. (2016). Lichens were collected using a knife or hammer and chisel and subsequently air-dried and stored in paper bags.
Specimens were observed and identified by the first author using an Olympus SZX7 stereomicroscope and an Olympus BX50 compound mic roscope with interference contrast, connected to a Nikon Coolpix digital ca mera. Sections were mounted in tap water, in which all measurements were also taken. The chemistry of selected specimens was investigated by thin-layer chromatography (Orange et al. 2001), using solvent A.
DNA analysis based on mitochondrial ribosomal small subunit (mtSSU) sequences was carried out for ten unidentifiable or provisionally identified specimens of good quality (indicated in Table 1). Although the nuclear ribosomal ITS region is the generally accepted fungal DNA barcode locus (Schoch et al. 2012), mtSSU was chosen since more mtSSU than ITS sequences have yet been published for several of the genera or families to which the respective specimens putatively belong.
Genomic DNA was extracted using the NucleoMag 96 Plant kit (Macherey-Nagel) on the KingFisher Flex Purification System (ThermoFisher Scientific). The mtSSU region was PCR-amplified following Zoller et al. (1999) in terms of primers (mrSSU1/ mrSSU3R) and the PCR protocol. PCR products were purified and sequenced at Base-Clear B.V. (www.baseclear.com) using the amplification primers. Sequences were assembled and edited using Geneious v8.1.8 (Biomatters Ltd.) and subjected to a BLAST search (Altschul et al. 1990) against the GenBank database (megablast; considering, where possible, BLAST results with E value of 0.0 and query cover >90 %). Sequences are available in GenBank under accession numbers MH028639−MH028646.
To compile an updated list of the lichens of St. Eustatius, literature and internet sources were exhaustively consulted for previous reports and previous collectors were contacted for additional information.

Results and discussion
In total, 126 lichen species (and one identifiable lichenicolous fungus) were found in 243 collections (Table 1). The vast majority (113 species) could be identified to species level based on morphological and chemical characters, even though no identification book exists for any region nearby. However, many species have been described from other islands in the Caribbean, which can be expected to have many species in common. These were often already described in the 19 th century and partly never studied again, but illustrations of their types are increasingly available. The authors also had access to various unpublished sources, such as the unpublished keys, descriptions and specimen citation (by H. Sipman) that was the basis of the internet checklist of St. Eustatius lichens and keys to the lichens from Puerto Rico (Harris 1989) and Guadeloupe (Øvstedal 2010), the latter with many illustrations of type and other specimens.  Table 2. Plots H1 and H2 are situated in the Northern Hills, M2−M9 on and around the volcano The Quill in the southern part of the island and U1 in the central urban area. Substrates occupied by each taxon are indicated per plot; b: bark, k: limestone, l: leaves, r: siliceous rock, s: soil, w: wood. Asterisks indicate first records for St. Eustatius (asterisks in brackets indicate additional taxa that are not yet identified to species level). Black dots (•) indicate specimens from which DNA was extracted. Somewhat to the authors' surprise, as many as 54 (almost 50 %) of the identified species are new records for St. Eustatius. This includes mostly relatively common and widespread tropical or Neotropical species, but also some rare species, notably Staurolemma dussii (Vain.) P.M. Jørg. & Henssen, which was so far only known from its type from Guadeloupe. Furthermore, it is remarkable that Cresponea flava (Vain.) Egea & Torrente was found on siliceous rock. The presence of so many additional species within the limited surface area of the plots, totalling 6875 m 2 (0.03% of the total island surface), suggests that the exploration of the lichen flora of St. Eustatius has not yet been exhaustive. However, no clearly undescribed species were found in the material and the number of species described based on material from St. Eustatius remains low with two, viz. Stirtonia neotropica (Aptroot 2009) and Syncesia subintegra (Sipman 2009).
Several specimens could not be identified with certainty in the present material but represent additional species (and in several cases additional genera). These are, for instance, Lichinaceae and Verrucariaceae, of which the taxonomy of the tropical taxa is incompletely known. Rather than describing them as new, they were listed with the name of the species that is morphologically most similar, preceded by "cf ". The BLAST results from the mtSSU sequences obtained from eight of these specimens in most cases allowed preliminary insights into their phylogenetic position.
The sequence of the Anisomeridium specimen with only conidia from St. Eustatius receives the highest BLAST hits with other representatives of the Monoblastiaceae in Nelsen et al. (2009Nelsen et al. ( , 2011 Both the apotheciate and sorediate Bacidia specimens are closest to sequences of species of the Toninia-Bacidia p.p. clade in Miadlikowska et al. (2014), the former to Toninia sedifolia (Scop.) Timdal (94 % identity) and the latter to Bacidia californica S. Ekman and B. phacodes Körb. (88−89 % identity), respectively. Consequently, they do not belong to Bacidia s.str., which forms a separate clade (including the type species, B. rosella (Pers.) De Not.) in Miadlikowska et al. (2014).
In the Verrucariaceae, Verrucaria was resolved as polyphyletic and Thelidium mixed with Polyblastia, Staurothele p.p. and Verrucaria p.p. (Polyblastia clade) in molecular phylogenetic reconstructions (Gueidan et al. 2007, Muggia et al. 2010, Thüs et al. 2011. The sequence of the Thelidium cf. decipiens specimen from St. Eustatius, how- type, but some are less specialised. Also, there is a marked difference between the lichens of the different plots and the three main areas on St. Eustatius (Northern Hills, central urban area, The Quill). However, the authors refrain from performing statistical comparisons of the lichen diversity between plots, since the number of plots per main area differs and is still low and the sampling strategy was devised by specialists of other organism groups. Nevertheless, the lichen data will be useful for an island-wide, plot-based comparison of diversity amongst all organism groups sampled during the 2015 inventory.
In Table 2, an updated checklist is presented of the lichens of St. Eustatius, citing only identified species, but based on all available sources and with their taxonomy (nomenclature and sometimes species concept) updated where necessary. According to this list, a total of 263 species are currently known from St. Eustatius. As a side effect of revising the existing records, one record becomes questionable, viz. Myriostigma candidum Kremp., which is not known from the Neotropics. It is intended to continue the exploration of the lichens of this island in the near future.