Limonium species endemic to Zakynthos Island 591
was immediately placed in silica gel to dry. To ensure sam- pling of separate individuals, material was only collected from plants at least 5 m apart. Total genomic DNA was ex- tracted from 20 mg of dried leaf samples using the NucleoSpin Plant II kit (Macherey-Nagel, Germany) fol- lowing the manufacturer’s instructions. The quantity and quality of the extracted DNA were assessed by agarose gel electrophoresis and spectrophotometer. We stored
DNA samples at −20 °C until used. Of the eight microsatellite markers initially selected for genotyping (Supplementary Table 1), five (Ln39,Ln68, Ln115,Ln152,Ld423) that showed correct amplification pat- tern by polymerase chain reaction (PCR) testing were se- lected and genotyped. Multiplex PCR reactions were carried out in 96-well plates containing c. 10 ng of template DNA, 0.2 μM forward and reverse primers, 1.5 mM MgCl2, 0.2mMdNTPs, 1 × KAPATaq buffer and 1Uof KAPATaq DNA Polymerase (Kapa Biosystems, USA). Cycling con- ditions consisted of an initial 94 °C denaturation step for 5 min, followed by 30 cycles of 40 sat 94 °C, 50 sat 54/64 °C (depending on the primer pairs used) and 50 sat 72 °C, with a final extension at 72 °C for 7 min. PCR products were separated on SeqStudio Genetic Analyzer (Applied Biosystems, USA). Allele sizes were determined using STRand 2.4.110 (Toonen & Hughes, 2001). We used GenAlEx 6.5 (Peakall & Smouse, 2012) and GENEPOP 4.7.5 (Raymond & Rousset, 1995; Rousset, 2008) to calculate the numbers of alleles (Na) and effective alleles (Ne), Shannon’s information index (I), observed (Ho) and expected (He) heterozygosity, total mean fixation index (FST), inbreeding coefficient (Fis) and gene flow (Nm).
Results Distribution
Limonium korakonisicum is currently known only from its type locality in Korakonisi, south-west Zakynthos (Fig. 1). Its EOO and AOO are 4 km2,and itslocal EOOis 463 m2 (Tables 1 & 2; values of local EOO for the three species are for 2018). The population of L. phitosianumcomprises 14 subpopu-
lations (Table 1, Fig. 2). We discovered several new subpo- pulations (Lp2,Lp4,Lp9,Lp10,Lp12,Lp13,Lp14), expanding the known range of this species. Habitat loss has occurred, however, as a result of anthropogenic activities, including the construction of a port that led to the extinction of a subpopulation in Aghios Nikolaos (Volimes). The EOO of L. phitosianum is 460 km2, the AOO is 60 km2 and the local EOO is 83,443 m2 (Fig. 2). We confirmed L. zacynthium at all of its known local-
ities except its locus classicus on Keri Lake beach, which has suffered habitat destruction from a tourism-related
FIG. 1 Distribution of Limonium korakonisicum and Limonium zacynthium, their area of occupancy (i.e. the number of occupied 2 × 2 km grid cells), and the estimated extent of occurrence (EOO) of L. zacynthium. Numbers indicate subpopulations (Lz1, etc.; Table 1).
development. We discovered one new subpopulation, Lz3 (Pelouzo islet). The species has three large subpopulations (Lz1,Lz2,Lz3) but subpopulations Lz4 and Lz5 comprise only 2–5 individuals. The EOO of L. zacynthium is 93 km2,the AOOis 28 km2 and the local EOO is 9,036 m2 (Fig. 1). Re-evaluation of the range of the three species in 2023
revealed no substantial change, except for a decrease in the local EOO of subpopulation Lp11, probably as a result of new recreational facilities in Porto Roxa. The EOO and AOO of L. phitosianum were not affected, however.
Population size
The population size of L. korakonisicum remained relatively stable during the monitoring period. Limonium phitosia- num showed varying trends, with some subpopulations decreasing and others increasing in size. The population of L. zacynthium significantly declined in 2018, primarily because of reductions in the Lz1 subpopulation (Table 2). By 2023, the population size of all three species had de- creased (Table 2). Observations of developmental stages were consistent across all studied species, with a predomi- nance of reproductive individuals in all subpopulations and with seedlings comprising only a small proportion of individuals (Supplementary Table 2).
Oryx, 2024, 58(5), 587–599 © The Author(s), 2024. Published by Cambridge University Press on behalf of Fauna & Flora International doi:10.1017/S0030605324000140
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