Recent Efforts to Recover Armeria Berlengensis, an Endemic Species from Berlengas Archipelago, Portugal

Article Recent Efforts to Recover Armeria berlengensis, an Endemic Species from Berlengas Archipelago, Portugal

Teresa Mouga 1,* , Susana Mendes 1 , Inês Franco 2, Ana Isabel Fagundes 3, Nuno Oliveira 3 , Paulo Crisóstomo 4, Lurdes Morais 4 and Clélia Afonso 1

1 MARE—Marine and Environmental Sciences Centre, ESTM, Polytechnic of Leiria, Edifício CETEMARES, Av. Porto de Pesca, 2520-641 Peniche, Portugal; [email protected] (S.M.); [email protected] (C.A.) 2 ESTM, Polytechnic of Leiria, 2520-641 Peniche, Portugal; [email protected] 3 SPEA—Sociedade Portuguesa para o Estudo das Aves, Avenida Columbano Bordalo Pinheiro, 87 – 3◦ Andar, 1070-062 Lisboa, Portugal; [email protected] (A.I.F.); [email protected] (N.O.) 4 ICNF—Instituto da Conservação da Natureza e das Florestas, Reserva Natural das Berlengas. Av. Mariano Calado, 57, 2520-224 Peniche, Portugal; [email protected] (P.C.); [email protected] (L.M.) * Correspondence: [email protected]; Tel.: 351-262-783-607

Abstract: Berlengas archipelago is a UNESCO world heritage site and the only location where Armeria berlengensis is found. This species faces various threats, namely, human disturbance, the presence of Carpobrotus edulis, yellow-legged gull, common-rabbit, and black-rat populations. Thus, exclusion areas were installed, which blocked the access of most Gulls, aiming to promote the recovery of A. berlengensis. Additionally, rabbits and rats were removed from the island. After six years of surveys, there has been an increase in the number of individuals of A. berlengensis in the exclusion areas, and a clear shift in the size structure of the A. berlengensis population. Significant changes in the height and diameter of the individuals were also noted. These findings indicate that the population of Citation: Mouga, T.; Mendes, S.; A. berlengensis is changing and becoming a healthier population. Principal component analysis results Franco, I.; Fagundes, A.I.; Oliveira, show a straightforward dissimilarity between the areas with A. berlengensis and those without the N.; Crisóstomo, P.; Morais, L.; Afonso, species and allowed the clustering of two groups: the rupicolous species and the nitrophilous species. C. Recent Efforts to Recover Armeria A. berlengensis produces few seeds (seed set 3.4%), which raises concern regarding the long-term berlengensis, an Endemic Species from Berlengas Archipelago, Portugal. survival of the species. Thus, further conservation efforts must be implemented, such as the control Plants 2021, 10, 498. https://doi.org/ of invasive species, gulls, and ruderals, to allow for the recovery of A. berlengensis. 10.3390/plants10030498 Keywords: conservation; endangered species; flora; island; rupicolous species; threats Academic Editor: Pilar Catalan

Received: 15 February 2021 Accepted: 3 March 2021 1. Introduction Published: 7 March 2021 The Berlengas archipelago was firstly classified as a natural reserve in 1981. In 1998, this area was classified as a marine reserve area, and its territory increased to its present size Publisher’s Note: MDPI stays neutral (terrestrial surface 725.6 ha, marine area of 17,776.7 ha). In 2011, the Berlengas became part with regard to jurisdictional claims in of the list of UNESCO world heritage sites, which aim to preserve representative terrestrial published maps and institutional affil- and marine ecosystems of the Portuguese coast. The Berlengas archipelago hosts several iations. important species and has other interesting features, which are relevant in the national and international context. These unique features of the archipelago are due to its insular nature, geological characteristics, geographical location, and climate, along with historic low levels of human interference. All together, these characteristics have contributed to the Copyright: © 2021 by the authors. preservation and speciation of the terrestrial and marine flora and fauna [1,2]. Licensee MDPI, Basel, Switzerland. Oceanic islands are known for their high levels of plant diversity, due to disjunct This article is an open access article geographical distribution that leads to speciation [3,4]. The main factors contributing to distributed under the terms and genetic speciation include the creation of a barrier within a previously widley distributed conditions of the Creative Commons taxon [5] and the limited dispersal of seeds, which favors genetic differenciation and, thus, Attribution (CC BY) license (https:// fosters rapid speciation [6]. In the Berlengas archipelago, this speciation was due to island creativecommons.org/licenses/by/ 4.0/). isolation from the continent that occurred after the Wurm Glatiation, ca. 18,000 B.C. [7].

Plants 2021, 10, 498. https://doi.org/10.3390/plants10030498 https://www.mdpi.com/journal/plants Plants 2021, 10, 498 2 of 19

Owing to the insular isolation, and to very harsh climatic and edaphic conditions, most of the botanical species are rupicolous aero-halophytic plants that can tolerate the presence of salt and action of strong winds, simultaneously. The terrestrial flora evolved differently from the mainland species, as these plants struggle to survive in extreme summer dryness, very thin soils, salty environments, and strong regular north-westerly and intense south- westerly winds. Most of the plants are thicker and smaller in size than their continental counterparts, with smaller leaves, which prevent wind damage and excess evaporation. These harsh conditions led to speciation processes that differentiated three endemic species of great conservation value: Armeria berlengensis Daveau, Herniaria berlengiana (Chaudhri) Franco and Pulicaria microcephala Lange [8–10]. As most island endemic plants, these species have a small number of individuals and a low distribution, thus, they are particularly susceptible to extinction [4]. Plant survival and population fitness vary according to environmental factors and to human interference [11]. Endemic island plant species from the Mediterranean area are generally threatened by urbanization, touristic activities, fire, invasive alien species, changes in agriculture practices and collecting pressure [4]. The endemic and halophytic flora of the Berlengas Archipelago is also threatened by these factors, as observed in many other European coastal territories which host threatened endemics with very limited areas of distribution [4,11–14]. First, increasing tourism activities during recent decades have led to the disturbance of the ground in recreational areas. Additionally, the introduction—in the late 1950s—of the African species Carpobrotus edulis (L.) N.E.Br., in an attempt to reduce rockfalls in recreational areas has also led to declines in native species. This invasive species spread out over the cliffs and hillsides [15], reaching a total area of 38,533 m2 in Berlenga Grande, which represented around 37% of its terrestrial area [16]. Other species, such as the ruderals Calendula suffruticosa subsp. algarbiensis (Boiss.) Nyman, Cerastium glomeratum Thuill., Echium rosulatum Lange, Fumaria muralis Sond. ex Koch, and Hyoscyamus albus L., Linaria amethystea subsp. multipunctata (Brot.) Chater & D.A. Webb, Nicotiana glauca Graham, tend to flourish in more disturbed areas, namely, where human intrusion is stronger, such as along paths and construction sites [17]. Furthermore, the increase in the amount of phosphorus and nitrogen produced by the large population of yellow-legged gull (Larus michahellis) that breeds on the Berlengas Archipelago has caused significant soil changes, namely, increases in water and nutrient retention, which increases organic matter, and decreases the soil pH [18]. These soil changes have considerably altered the natural floristic composition, promoting the emergence of annual nitrophilous vegetation and the decline of autochthonous perennial species [17,19–22]. Finally, introduced herbivores—the black-rat (Rattus rattus) and common-rabbit (Oryctolagus cuniculus)—have also played a role in decreasing the natural vegetation. It is well known that extensive grazing increases open land habitats, thus, improving grasslands and causing the decline of shrublands [23]. Grazing on perennial plants by rabbits, which also profusely excavate the thin soil, has put extensive pressure on A. berlengensis [1]. As for rats, these are omnivores that feed on vertebrates, invertebrates, fungi and plants, but not on A. berlengensis, as stated by Nascimento, T. [24]. Yet, Carpobrotus spp. fruits have been found to be eaten and dispersed by both rabbits and rats, thus contributing to C. edulis success [25]. Additionally, gull- derived resources are significant for both rodent species, and these have altered their growth rates, reproductive output, and population densities [26]. In short, the species A. berlengensis has changed in status from vulnerable [15] to critically endangered, according to the International Union for Conservation of Nature (IUCN)threat classification scheme, due to the presence of C. edulis, herbivores, and L. michahellis that nest over the most mature plants [27,28]. A. berlengengis is registered in Annex II of the Habitats Directive, due to their conservation relevance [28,29]. Therefore, to preserve important habitats and species, the control of L. michahellis has been undertaken for more than twenty years—through egg removal—to control the size of the nesting population on the island. Additionally, between 2016 and 2018, black-rat eradication campaigns were implemented and in 2017 and 2018, rabbits Plants 2021, 10, x FOR PEER REVIEW 3 of 19

100 of L. michahellis has been undertaken for more than twenty years—through egg removal— 101 to control the size of the nesting population on the island. Additionally, between 2016 and 102 2018, black-rat eradication campaigns were implemented and in 2017 and 2018, rabbits 103 were also eliminated from the Berlengas main island. In addition, in 2014, conservation 104 actions commenced that permitted the removal of more than 90% of C. edulis from the 105 main island [16]. Finally, in May 2015, several areas with metal structures and fishing lines Plants 2021, 10, 498 3 of 19 106 were installed (exclusion areas), which blocked the access of most gulls. The vegetation of 107 these areas has been seasonally monitored between 2015 and 2020 to assess vegetation 108 evolution. Spring data were analyzed during the study period to evaluate vegetation var- were also eliminated from the Berlengas main island. In addition, in 2014, conservation 109 iationactions commencedduring the that most permitted productive the removal season of more on thanthe 90%island. of C. edulisSeed fromset was the main also measured to 110 betterisland [ 16understand]. Finally, in Maythe natural 2015, several reproduction areas with metal rates structures of A. berlengensis and ﬁshing. lines were 111 installedThis (exclusion study, areas),therefore, which aimed blocked to the determine access of most the gulls. effectiveness The vegetation of ofvarious these conservation 112 strategiesareas has been on seasonally the population monitored of between A. berlengensis 2015 and 2020. As to such, assess we vegetation assessed evolution. the effectiveness of Spring data were analyzed during the study period to evaluate vegetation variation during 113 the establishment of exclusion zones on the growth promotion of A. berlengensis, on the the most productive season on the island. Seed set was also measured to better understand 114 growththe natural of reproduction the other rupicolous rates of A. berlengensis species,. and on the growth of nitrophilous species. The 115 heightThis and study, diameter therefore, of aimed A. toberlengensis determine thewas effectiveness also determined of various conservationannually to evaluate the 116 growthstrategies of on the the populationindividuals of A.and berlengensis the size. Asstructure such, we of assessed the A. the berlengensis effectiveness population of in the the establishment of exclusion zones on the growth promotion of A. berlengensis, on the 117 studied areas. Moreover, we evaluated the number of seeds produced by A. berlengensis— growth of the other rupicolous species, and on the growth of nitrophilous species. The 118 byheight seed and cluster—to diameter of A. estimate berlengensis thewas seed also set determined of the species, annually toin evaluate attempt the to growth understand the re- 119 coveryof the individuals capacity andof the the species. size structure of the A. berlengensis population in the studied areas. Moreover, we evaluated the number of seeds produced by A. berlengensis—by seed 120 2.cluster—to Results estimate the seed set of the species, in attempt to understand the recovery capacity of the species. 121 2.1. Armeria Berlengensis Surveys 2. Results 122 After six years of surveying the exclusion and control areas, there is clear evidence of 2.1. Armeria Berlengensis Surveys 123 the nonexistence of A. berlengensis organisms in all the areas where it was initially absent After six years of surveying the exclusion and control areas, there is clear evidence of 124 (allthe nonexistenceB areas), except of A. berlengensisfor one deadorganisms adult inindividu all the areasal observed where it was in 2015 initially (height absent 30–40 cm). This 125 individual(all B areas), exceptwas excluded for one dead from adult the individual following observed surveys. in 2015 (height 30–40 cm). This 126 individualYet, in was the excluded areas fromwere the A. following berlengensis surveys. initially existed, a significant difference was no- 127 ticeableYet, in(t-student the areas test, were pA.-value berlengensis = 0.013)initially between existed, the a number signiﬁcant of difference individuals was growing in the noticeable (t-student test, p-value = 0.013) between the number of individuals growing in 128 exclusion areas and the control areas (Figure 1), demonstrating the heterogeneity of the the exclusion areas and the control areas (Figure1), demonstrating the heterogeneity of 129 data.the data.

130 Figure 1. Mean number of individuals of A. berlengensis in the exclusion areas A(1–3) “structure + 131 Figure 1. Mean number of individuals of A. berlengensis in the exclusion areas A(1–3) “structure + Armeria” and the control areas A(4–6) “no structure + Armeria”. Results are presented as mean ± 95% 132 ArmeriaConﬁdence” and Interval the (CI).control areas A(4–6) “no structure + Armeria”. Results are presented as mean ± 133 95% Confidence Interval (CI). When only analyzing the total number of individuals of A. berlengensis in the different 134 areas,When during only the Spring analyzing (2015 to the 2020), total when number the species of individuals grows and blossoms, of A. berlengensis we can see in the different that this value ranged from 85 to 201 in the exclusion areas A(1–3) and from ﬁve to 17 for 135 areas, during the Spring (2015 to 2020), when the species grows and blossoms, we can see

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Plants 2021, 10, x FOR PEER REVIEW 4 of 19 the control areas A(4–6). Thus, a well-deﬁned trend of growth in the number of individuals can be seen, mainly in 2020 (Table1).

136 Tablethat this 1. Total value number ranged of A. from berlengensis 85 to individuals201 in the in exclusion the exclusion areas areas A(1–3) A(1–3) and “Structure from + fiveArmeria to 17”, and for 137 thein the control control areas areas A(4–6). A(4–6)”No Thus, structure a well-defined + Armeria” andtrend B(4–6) of growth “No structure in the +number no Armeria of individu-”, counted 138 alsbetween can be the seen, spring mainly of 2015 in (“spring15”) 2020 (Table and 1). the spring of 2020 (“spring20”).

139 TableTotal 1. Total Number number of of A. berlengensis individuals in the exclusion areas A(1–3) “Structure + Ar- Spring15 Spring16 Spring17 Spring18 Spring19 Spring20 140 meria”,Individuals and in the control areas A(4–6)”No structure + Armeria” and B(4–6) “No structure + no Ar- 141 meria”, counted between the spring of 2015 (“spring15”) and the spring of 2020 (“spring20”). A(1–3): Structure + 85 88 90 96 118 201 Total numberArmeria of individuals spring15 spring16 spring17 spring18 spring19 spring20 A(4–6): No structure + A(1–3): Structure + Armeria 785 688 890 96 5 1186 201 17 Armeria A(4–6):No structure + Armeria 7 6 8 5 6 17 B(4–6): No strucuture B(4–6):No strucuture + no 1 0 0 0 0 0 + no Armeria 1 0 0 0 0 0 Armeria Total 93 94 98 101 124 218 Total 93 94 98 101 124 218

142 When assessing assessing the the differences differences between between the the sampling sampling periods, periods, for each for eachtype of type struc- of 143 turestructure (namely, (namely, exclusion exclusion areas areasA(1–3) A(1–3) “structure “structure + Armeria + Armeria” and the” and control the controlareas A(4–6) areas 144 A(4–6)“no structure“no structure + Armeria + Armeria”), it was”), observed it was observed that these that are these not statistically are not statistically significant significant (ANO- 145 (ANOVAVAA(1–3), pA(1–3)-value, p >0.05-value and >0.05 Kruskal–Wallis and Kruskal–WallisA(1–6), p-valueA(1–6), >p -value0.05). > 0.05). 146 Additionally, when comparing the type of structure (namely, exclusion areas A(1–3) 147 “structure“structure ++ Armeria” and the control areas A(4–6) “no structure + Armeria”), for each each sam- sam- 148 pling periodperiod therethere werewere no no statistically statistically significant significant differences differences for for the the number number of individualsof individ- 149 ualsof A. of berlengensis A. berlengensis(t-student (t-student test, test,p-value p-value > 0.05 > 0.05 for for all all cases). cases). When When analyzing analyzing thethe datadata 150 globally, that is, whenwhen comparingcomparing the number of individuals of A. berlengensis for the six 151 sampling periods, the results also show the same pattern, that is, no statistically statistically significant significant 152 differences (ANOVA, pp-value-value >> 0.05)0.05) (Figure(Figure2 2).).

153 154 Figure 2. Mean number of individuals of A. berlengensis growing in all the areas analyzed,analyzed, counted between the spring of 155 2015 and the springspring ofof 2020.2020. Results areare presentedpresented asas meanmean valuesvalues ±± SD.SD.

Between 2015 and 2020 there was a clear increase in the number of plantlets, mainly 156 Between 2015 and 2020 there was a clear increase in the number of plantlets, mainly in 2019 for the exclusion area A(1–3) (Figure3a) and in 2020 for both exclusion areas A(1–3) 157 in 2019 for the exclusion area A(1–3) (Figure 3a) and in 2020 for both exclusion areas A(1– and control areas A(1–6) (Figure3b). It was possible to observe an increasing trend in 158 3) and control areas A(1–6) (Figure 3b). It was possible to observe an increasing trend in 159 the number of plantlets, mostly after 2019, when the total number of plantlets surpassed 160 the number of juvenile plants, for the first time, during our study. The other categories 161 remained rather stable, showing minor fluctuations between the years. Moreover, the very

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the number of plantlets, mostly after 2019, when the total number of plantlets surpassed Plants 2021, 10, x FOR PEER REVIEW 5 of 19 the number of juvenile plants, for the ﬁrst time, during our study. The other categories remained rather stable, showing minor ﬂuctuations between the years. Moreover, the very low number of large plants (>41 cm) in all the areas surveyed was very noticeable. 162 lowHowever, number these of large plants plants are (>41 oflarge cm) in dimensions all the areas and surveyed take up was considerable very noticeable. space How- in the 163 ever,study these area. plants are of large dimensions and take up considerable space in the study 164 area.

(a)

(b)

165 FigureFigure 3. 3. VariationVariation of ofthe the size size structure structure of A. of berlengensisA. berlengensis populationpopulation in the in studied the studied areas areas between between the Spring the Spring of 2015 of and 2015 166 the Spring of 2020: (a) in the exclusion areas A(1–3) “Structure + Armeria” (b) in the control areas A(4–6),”No structure + and the Spring of 2020: (a) in the exclusion areas A(1–3) “Structure + Armeria”(b) in the control areas A(4–6),”No 167 Armeria”. structure + Armeria”.

168 RegardingRegarding the the mean mean height height and and mean mean diameter diameter of of the the plants, plants, as as expected, expected, we we can can see see 169 a aclear clear correlation correlation between between both both measures, measures, fo forr all all the years and both exclusionexclusion areaarea A(1–3)A(1– 170 3) and control A(1–6) (R2 2A(1–3) “structure + Armeria” = 0.688, p-value < 0.01 and R22A(4–6) “no and control A(1–6) (R A(1–3) “structure + Armeria” = 0.688, p-value < 0.01 and R A(4–6) “no 171 structurestructure + +ArmeriaArmeria”” = =0.760, 0.760, p-valuep-value < <0.01; 0.01; Figure Figure 4),4), indicating indicating that that the the increase increase in in height height 172 isis accompanied accompanied by by an an increase increase in in width. width.

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173 Figure 4. Pearson’s correlation between height (h) and diameter (D) of the individuals of A. berlengensis, for all the 174 Figure 4. Pearson’s correlation between height (h) and diameter (D) of the individuals of A. berlengensis, for all the indi- 175 individualsviduals measured measured in the in exclus the exclusionion areas areas A(1–3) A(1–3) “Structure “Structure + Armeria + Armeria” and” the and control the control areas A(1–6) areas A(1–6) “No structure “No structure + Arme- + 176 Armeriaria”, between”, between the spring the spring of 2015 of 2015(“15”) (“15”) and the and spring the spring of 2020 of 2020(“20”). (“20”).

A. berlengensis 177 As for the height of A. berlengensis, it varied between 11.05 cm in 2020, corresponding mainly to plantlets and small adults, for the exclusion areas A(1–3) and 25.83 cm in 2019, 178 mainly to plantlets and small adults, for the exclusion areas A(1–3) and 25.83 cm in 2019, for the control areas A(4–6), corresponding to larger adult plants (Figure5a). The mean 179 for the control areas A(4–6), corresponding to larger adult plants (Figure 5a). The mean values of the plants in the exclusion area A(1–3) was 15.54 cm and in the control areas 180 values of the plants in the exclusion area A(1–3) was 15.54 cm and in the control areas was was 19.39 cm. The diameter of A. berlengensis varied from 18.7 cm in 2020, corresponding 181 19.39 cm. The diameter of A. berlengensis varied from 18.7 cm in 2020, corresponding mainly to plantlets and small adults, for the exclusion areas A(1–3) to 42.50 cm in 2017, for 182 mainly to plantlets and small adults, for the exclusion areas A(1–3) to 42.50 cm in 2017, for the control areas (A4–6), also corresponding to larger adult plants. The mean diameter of 183 the control areas (A4–6), also corresponding to larger adult plants. The mean diameter of the plants in the exclusion areas A(1–3) was 26.19 cm and in the control areas A(1–6) was 184 the plants in the exclusion areas A(1–3) was 26.19 cm and in the control areas A(1–6) was 32.25 cm (Figure5b). 185 32.25 cm (Figure 5b). Additionally, when comparing the height between the six sampling periods (sepa- 186 Additionally, when comparing the height between the six sampling periods (sepa- rately by type of area, namely, for the exclusion areas A(1–3) “Structure + Armeria” and 187 rately by type of area, namely, for the exclusion areas A(1–3) “Structure + Armeria” and the control areas A(1–6) “No structure + Armeria”), the results did not show statistically 188 the control areas A(1–6) “No structure + Armeria”), the results did not show statistically significant differences, for the control areas A(1–6) “No structure + Armeria” (ANOVA, 189 significant differences, for the control areas A(1–6) “No structure + Armeria” (ANOVA, p- p-value > 0.05). However, when the comparison was made for the exclusion areas A(1–3) 190 “Structurevalue > 0.05). + Armeria However,”, the when results the showedcomparison statistically was made significant for the differencesexclusion areas when A(1–3) com- 191 paring“Structure the sampling+ Armeria period”, the results (Kruskal–Wallis, showed statisticallyp-value < 0.001;significant Figure differences5a). More specifically,when com- 192 differencesparing the sampling can be observed period (Kruskal–Wallis, when comparing p the-value spring < 0.001; of 2020 Figure with 5a). all More the others specifically, (Dunn, 193 pdifferences-value < 0.001 can ),be as observed well as the when Spring comparing of 2019 withthe spring that of of 2017 2020 (Dunn, with allp-value the others = 0.045) (Dunn, and 194 2015p-value (Dunn, < 0.001;),p-value as well = 0.003) as the (Figure Spri5nga). of 2019 with that of 2017 (Dunn, p-value = 0.045) 195 and 2015For the(Dunn, diameter, p-value the = 0.003) results (Figure follow 5a). a similar pattern, as they showed statistically 196 significantFor the differences diameter, whenthe results comparing follow thea similar sampling pattern, periods, as they only showed for the statistically exclusion areas sig- 197 nificant differences when comparing the sampling periods, only for the exclusion areas (Kruskal–WallisA(1–3) “Structure + Armeria”, p-value < 0.001; Figure5b) and Kruskal– 198 (Kruskal–WallisA(1–3) “Structure + Armeria”, p-value < 0.001; Figure 5b) and Kruskal– WallisA(1–6) “No structure + Armeria”, p-value > 0.001). In particular, for the exclusion areas, 199 theWallis observedA(1–6) “No differences structure were + Armeria between”, p the-value Spring > 0.001). 2020 and In particular, all others (Dunn, for thep exclusion-value ≤ 0.001areas,), 200 asthe well observed as between differences Spring were 2019 andbetween Spring the 2016 Spring to 2018 2020 (Dunn, and allp-value others < (Dunn, 0.05) and, p-valuefinally, ≤ 201 between0.001;), as Spring well as 2015 between and alsoSpring Spring 2019 2016 and toSpring 2018 2016 (Dunn, to 2018p-value (Dunn, < 0.05) p-value (Figure < 0.05)5b). and, 202 finally, between Spring 2015 and also Spring 2016 to 2018 (Dunn, p-value < 0.05) (Figure 203 5b).

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(a)

(b)

204 Figure 5. FigureMean (a) 5. heightMean of(a) theheight plants of of the A. berlengensis plants of A. present berlengensis in the presentexclusion in areas the exclusion A(1–3) areas A(1–3) 205 “Structure“Structure + Armeria +” Armeriaand in the” and control in the areas control A(1–6) areas “No A(1–6) Structure “No Structure+ Armeria +” andArmeria (b) diameter” and (b) ofdiameter of 206 the plants of A. berlengensis present in the exclusion areas A(1–3) “Structure + Armeria” and in the the plants of A. berlengensis present in the exclusion areas A(1–3) “Structure + Armeria” and in the 207 control areas A(1–6) “No Structure + Armeria”, between Spring 2015 and Spring 2020, in centime- control areas A(1–6) “No Structure + Armeria”, between Spring 2015 and Spring 2020, in centimeters. 208 ters. Results are presented as mean values ±SD. Symbols above bars represent significant statistical ± 209 differencesResults between are medium presented values as mean (* between values 2020SD. and Symbols all the other above years; bars # represent between 2019 signiﬁcant and statistical 210 other years;differences † between between 2015 and medium other years). values (* between 2020 and all the other years; # between 2019 and other years; † between 2015 and other years). 211 There is a decreasing trend in height and diameter that occurred mainly after 2018 in There is a decreasing trend in height and diameter that occurred mainly after 2018 212 the exclusion areas A(1–3) (Figure 6,c), but this trend did not occur in the control areas in the exclusion areas A(1–3) (Figure6,c), but this trend did not occur in the control areas 213 A(1–6), where these values are much more heterogeneous (Figure 6b,d). This is concomi- A(1–6), where these values are much more heterogeneous (Figure6b,d). This is concomitant 214 tant with the increase in the number of individuals previously discussed (Figure 3a). with the increase in the number of individuals previously discussed (Figure3a). 215 It is also worth mentioning that there are many outliers in the exclusion areas A(1–3) It is also worth mentioning that there are many outliers in the exclusion areas A(1–3) all 216 all corresponding to large adult plants, presenting greater height and/or larger diameter corresponding to large adult plants, presenting greater height and/or larger diameter than 217 than the median values. These represent the oldest individuals of the surveys, also men- the median values. These represent the oldest individuals of the surveys, also mentioned 218 tioned above in Figure 3. above in Figure3.

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(a) (b)

219 Figure 6. Boxplots for the medianFigure values 6. Boxplots of the height for the (h) median and the values diameter of the (D1), height in centimeters, (h) and the ofdiameter the vegetation (D1), in measured centimeters, of 220 between Spring 2015 and Springthe vegetation 2020. (a) Height measured of individuals between Spring of A. 2015 berlengensis and Springgrowing 2020. in (a the) Height exclusion of individuals areas A(1–3) of A. 221 berlengensis growing in the exclusion areas A(1–3) “structure + Armeria”. (b) Height of individuals “structure + Armeria”. (b) Height of individuals of A. berlengensis growing in the control areas A(4–6) “No structure + 222 of A. berlengensis growing in the control areas A(4–6) “No structure + Armeria”. (c) Diameter of Armeria”. (c) Diameter of individuals of A. berlengensis growing in the exclusion areas A(1–3) “No structure + Armeria”. (d) 223 individuals of A. berlengensis growing in the exclusion areas A(1–3) “No structure + Armeria”. (d) ◦ 224 Diameter of individuals of A.Diameter berlengensis of growingindividuals control of A. areas berlengensis A(4–6) “Nogrowing structure control + Armeria areas A(4–6)”. represent “No structure outliers + andArmeria * ”. ◦ 225 represent extreme outliers (thatrepresent is, even outliers more extreme and * repres outliers).ent extreme outliers (that is, even more extreme outliers).

226 2.2. PCA Analysis 2.2. PCA Analysis 227 The principal component analysis (PCA) was applied to the species coverage on dif- The principal component analysis (PCA) was applied to the species coverage on 228 ferent sampling periods. The PCA results showed that 51.6% of the total variance was different sampling periods. The PCA results showed that 51.6% of the total variance was 229 explainedexplained by by the the two two principal principal components. components. Additionally,Additionally, PC1 PC1 explained explained 36.7% 36.7% of of the the 230 variancevariance of of the the original original variables variables analyzed, analyzed, which which is theis the most most signiﬁcant significant response response regarding regard- 231 theing clear the separationclear separation between between the areas the with areas or withoutwith or A.without berlengensis A. berlengensis(Table2 and (Table Figure 2 7and) . 232 Figure 7). Table 2. Summary of PCA of the exclusion areas and the control areas. 233 Table 2. Summary of PCA of the exclusion areas and the control areas. 1 1 22 33 44 Total Total Variance Variance EigenvaluesEigenvalues 0.367 0.367 0.149 0.149 0.1190.119 0.0830.083 1 1 CumulativeCumulative percentage variance 36.7 51.6 63.5 71.8 -- percentageof species variance data 36.7 51.6 63.5 71.8 – of species data 234 Having clearly defined two clusters, the PCA results demonstrated that there is a 235 straightforward dissimilarity between the areas with A. berlengensis and the areas without 236 A. berlengensis (Figure 7). The areas A (A1 to A6) presented A. berlengensis during the entire

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Plants 2021, 10, 498 9 of 19 237 period of the study while the areas B (B1 to B6) failed to regain any individuals of A. 238 berlengensis, either in the exclusion or the control areas.

239 240 FigureFigure 7. 7.PCAPCA biplot biplot showing showing the the PC1, PC1, with with a a clear clear dissimilarity dissimilarity between between the the areas areas presenting presentingA. A. berlengensis berlengensis(A1 (A1 and and A2) A2) 241 andand those those without without any anyA. A. berlengensis berlengensis, (B1, (B1 and and B2) B2) for for all all the the sampling sampling period period (Spring (Spring 2015–Spring 2015–Spring 2020). 2020).

242 HavingRegarding clearly the species defined coverage two clusters, (%) pattern, the PCA in the results CP1 demonstrated positive axis, there that thereis a cluster is a 243 straightforwardthat includes A. dissimilarity berlengensis (sp1), between the themost areas characteristic with A. berlengensis rupicolousand species the areas on the without island, 244 A.alongside berlengensis Plantago(Figure coronopus7). The L. areas (sp7), A and (A1 Spergularia to A6) presented rupicola LebelA. berlengensis ex Le Jol. (sp9)during (Figure the 245 entire8). Additionally period of the related study to while A. berlengensis the areas, Bare (B1 the to different B6) failed species to regain from any the individualsPoaceae family of 246 A.(sp4) berlengensis and Polycarpon, either inalsinifolium the exclusion (Biv.) or DC. the (sp14). control areas. 247 RegardingIn the CP1 the negative species axis, coverage there (%)is anothe pattern,r cluster in the CP1which positive includes axis, the there ruderal is a clus-nitro- 248 terphilous that includes species, suchA. berlengensis as C. suffruticosa(sp1), subsp the most. algarbiensis characteristic (sp3) and rupicolous Echium speciesrosulatum on (sp5). the 249 island,Additionally, alongside thePlantago pattern coronopusobserved L.showed (sp7), that and Spergulariathese two species rupicola areLebel strongly ex Le correlated, Jol. (sp9) 250 (Figurewhich8 means) . Additionally that the increasing related to A.abundance berlengensis of,C. are suffruticosa the different subsp species. algarbiensis from the is Poaceae accom- 251 familypanied (sp4) by the and increasingPolycarpon abundance alsinifolium of(Biv.) E. rosulatum DC. (sp14)., and that the environmental condi- In the CP1 negative axis, there is another cluster which includes the ruderal ni- 252 tions that benefit one also benefit the other. Lobularia maritima (L.) Desv. subsp. maritima trophilous species, such as C. suffruticosa subsp. algarbiensis (sp3) and Echium rosulatum 253 (sp6) seems to be correlated with the former two species, but to a smaller extent, also being (sp5). Additionally, the pattern observed showed that these two species are strongly cor- 254 a ruderal species. Similarly, but less markedly, species such as Mercurialis ambigua L. related, which means that the increasing abundance of C. suffruticosa subsp. algarbiensis 255 (sp13), Erodium cicutarium (Cav.) Tourlet (sp10), Scrophularia sublyrata Brot. (sp11), and is accompanied by the increasing abundance of E. rosulatum, and that the environmental 256 Urtica membranacea Poir. (sp2), likewise showed an association with each other, all being conditions that benefit one also benefit the other. Lobularia maritima (L.) Desv. subsp. mar- 257 nitrophilous species. As for Atriplex prostrata Boucher ex DC. in Lam.t DC. (sp8), again a itima (sp6) seems to be correlated with the former two species, but to a smaller extent, also 258 nitrophilous species, this species is allocated along the second axis, meaning that its being a ruderal species. Similarly, but less markedly, species such as Mercurialis ambigua 259 growth is not affected by the distribution of rupicolous vegetation nor by the distribution L. (sp13), Erodium cicutarium (Cav.) Tourlet (sp10), Scrophularia sublyrata Brot. (sp11), and 260 of ruderals vegetation. Urtica membranacea Poir. (sp2), likewise showed an association with each other, all being nitrophilous species. As for Atriplex prostrata Boucher ex DC. in Lam.t DC. (sp8), again a nitrophilous species, this species is allocated along the second axis, meaning that its growth is not affected by the distribution of rupicolous vegetation nor by the distribution of ruderals vegetation. PlantsPlants 20212021, ,1010, ,x 498 FOR PEER REVIEW 1010 of of 19 19

261 262 FigureFigure 8. 8. PCAPCA biplot biplot for for the the distribution distribution of of the the most most abundant abundant species species of of Berlengas Berlengas island, island, for for all all the the sampling sampling period period 263 (Spring(Spring 2015–Spring 2015–Spring 2020). 2020).

264 TheThe remaining remaining species species showed showed a low a low relevance relevance behavior behavior since since their their variability variability is re- is 265 duced,reduced, as asvectors vectors are are closer closer to tothe the origin origin (Figure (Figure 8),8), showing showing a a slight slight contributory contributory power 266 forfor the the global global behavior behavior of of the the species species under under analysis. analysis.

267 2.3.2.3. Armeria Armeria berlengensis berlengensis Seed Seed Set Set 268 DuringDuring our our survey, survey, 98 98 fruit fruit clusters clusters were were analyzed, analyzed, with with a mean a mean number number of fruits of fruits per 269 clusterper cluster of 48.17. of 48.17. The results The results showed showed that the that total the number total number of seeds of produced seeds produced by A. ber- by 270 lengensisA. berlengensis is veryis low, very having low, having been collected been collected only 164 only seeds 164 seeds out of out 4721 of 4721calyxes. calyxes. There There is a 271 meanis a mean value value of 1.67 of 1.67seeds seeds per fruit per fruitcluster cluster and anda seed a seedset of set only of only3.47% 3.47% (Table (Table 3). 3).

272 Table 3. TotalTable number 3. Total of number A. berlengensis of A. berlengensis fruit clustersfruit opened, clusters and opened, seeds and recovered. seeds recovered.

DataData Analyzed Analyzed # # TotalTotal number number of fruits of fruits opened opened 4721 4721 Fruits Fruits MeanMean number number of fruits of fruits per cluster per cluster 48.17 48.17 TotalTotal number number of seeds of seeds collected collected 164 164 Seeds Mean number of seeds collected 1.67 Seeds 1.67 Mean numberSeed set of seeds collected 3.47% Seed set 3.47%

273 MoreMore specifically, speciﬁcally, by by observing observing 19 19 infructescences, infructescences, it it should should be be noted noted that that the the low low 274 seedseed set set observed observed (3.39%) (3.39%) was was mainly mainly due due to to th thee presence presence of of non-fertilized non-fertilized ovaries, ovaries, which which 275 accountedaccounted for for more more than than 50% 50% of ofthe the total total number number of flowers of ﬂowers analyzed analyzed (Table (Table 4). Addition-4). Addi- 276 ally,tionally, many many calyxes calyxes were empty were empty (30.53%), (30.53%), and more and than more 15% than were 15% found were that found were that already were 277 decaying.already decaying. 278

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Table 4. Seed set of Armeria berlengensis, percentage of empty calyxes, non-fertilized ovaries, and rotten calyxes observed in 19 infructescences observed in detail.

#% Number of fruit clusters analyzes 19 – Seed set 23 3.39 Empty calyxes 207 30.53 Non-fertilized ovaries 343 50.59 Rotten calyxes 105 15.49 Total number of calyxes 678 –

3. Discussion The results demonstrated that there is a rising number of individuals growing in all the studied areas A(1–6), but not where the species A. berlengensis was previously absent, such as in areas B, the exclusion areas B(1–3) or the control areas B(4–6). This means that the capacity of recruitment of the species is rather limited and that the presence of the structures installed in A(1–3) and B(1–3) is efficient in increasing the number of individuals in the vicinity of sites where there are individuals of A. berlengensis, but not beyond these A(1–3) areas. This can be explained by the fact that Armeria seeds are wind dispersed and the distance of seed transportation is low, as shown by Philipp et al. [30] for A. maritima (maximum distance of 3.5 m, mean distance 0.88 m). Therefore, during the six years of the surveys, we were unable to expand the distribution area of A. berlengensis. Additionally, it was clear that from the first years of the study and until 2018, the number of new individuals was very low, with a reduced number of individuals under 10 cm, when compared to the older individuals (especially young adults). Thus, for the first few years of the survey, the pyramid was truncated at its base, meaning that the reproduction and recruitment of the species seemed to be compromised [31], and so the survival status of the species seemed to be at risk. However, this pattern changed in the surveys from 2019 and 2020, when the recruitment of new plantlets augmented substantially, mainly in the exclusion areas A(1–3). Thus, the pyramid base also grew considerably, evolving into a much more equilibrated and healthy population, with a higher number of individuals belonging to younger classes, due to recruitment of new plants by germination [32]. The increase in the number of A. berlengensis plantlets in the control area is also noticeable, but to a much less extent than in the exclusion areas. Due to the windy habitat it is exposed to, A. berlengensis presents a prostrate, pulvini- form shape; therefore, the individuals are usually wider than they are tall, in line with most of the littoral shrubs [33]. Thus, the correlations found between diameter and height are to be expected, because as they grow, the plants become successively wider and taller. The significant differences found for the diameter and the height of the individuals of A. berlengensis in the exclusion areas A(1–3), can be explained by the recruitment of new individuals, occurring mainly in 2019 and 2020, as already discussed. New individuals are smaller in size and diameter, and thus, are responsible for the decreasing trend in diameter and height that occurred mainly after 2018. The significant differences observed both for diameter and height between 2020 and the other years is meaningful, for it is coincident with the increase in the number of new plantlets observed growing in the studied areas. The removal of herbivorous mammals from the island had a beneficial effect on the native vegetation recovery, as has been widely documented by many authors world- wide [34–41]. We believe that the growth of new plantlets is due to the removal of the mammals. The natural vegetation of Berlengas is chasmophytic, mainly composed of geophytes, hemicryptophytes, and chamaephytes, often covered by marine salt spray [42]. Therefore, the species A. berlengensis, N. bulbocodium, S. rupicola, S. sublyrata, and A. pachycharpa—all rupicolous species—should thrive on the island, mainly in undisturbed niches. Addition- ally, the association between A. berlengensis and S. rupicola has been acknowledged before, as they are specialized species that grow in granitic rock crevices and on cliffs, and they Plants 2021, 10, 498 12 of 19

are well adapted to harsh environmental conditions [33]. The PCA results reinforce the idea that the presence of A. berlengensis is accompanied by other species that are adapted to rocky environments and scarce soil. Such species include: Plantago coronopus, which is a characteristic species of dry grasslands with scarce soil; Spergularia rupicola, a species that grows in rocky places, near the shore [9]; Polycarpon alsinifolium, a species that grows in sandy habitats [43], and species from the Poaceae family. All these species are adapted to a thin soil layer and low nutrient content. Additionally, the exclusion area played no part in the recovery of the A. berlengensis population in areas where the species was already absent, as already stated, with the principal component PC1 clearly defining two very distinct areas. This means that the increase in the A. berlengensis population relies on the species current distribution area and may depend on the diminishing impact of the birds on the plateau. As stated, this may be due to the low distance seed dispersal. The differences observed between the exclusion and the control areas are due to a decrease in the inflow of gull droppings in the exclusion zones. These are known to be rich in nitrate and phosphate nutrients [29,44,45], which causes significant ruderalization of the vegetation [20,46]. Thus, the presence of some abundant ruderal species in the exclusion zones indicates that there is also disturbance in these areas, which reflects the presence of N-enriched soil, and/or the presence of gulls [21] before the installation of the structures. There is evidence that this part of the island (plateau) has been altered considerably due to the presence of gulls which have transported new annual and ruderal plant species to the island that thrive on this altered N-enriched soil [47]. Gulls must, therefore, be the main bottleneck to the growth of rupicolous vegetation on the island. Therefore, the presence of the structures on the exclusion zones, which discourage the occurrence of gulls, seems to be playing a significant role in the recovery of this type of vegetation. It is expected that in the coming years, in the exclusion zones, an increase in rupicolous vegetation will be observed as the N and P soil concentrations decrease. However, this will be a slow process. The PCA results also confirm that A. berlengensis and the other rupicolous species are on the CP1 positive axis, while C. suffruticosa subsp. algarbiensis and E. rosulatum are on the negative PC1 axis. These are both endemic species from the littoral area of the Iberian Peninsula, more robust, and well adapted to areas of greater soil accumulation, due to the gulls’ excrements, human activity and rock weathering [17,29]. The growth of rupicolous species occurs under the opposite environmental conditions to the growth of ruderal species. The ruderals, E. rosulatum and C. suffruticosa subsp. algarbiensis, presented higher cover percentages in places where A. berlengensis was absent (B areas, either exclusion or control zones), since they rapidly occupy the available N-enriched soil. Most of the species associated with these ruderals (e.g., L. maritima, M. ambigua, O. calendulae) also handle human interference well, and thus, prosper in disturbed soil [19]. Almost 40% of the biotopes of the Berlengas archipelago are from deep soil vegetation and only 16% belong to species that are adapted to crevices and thin soil layers. Additionally, it is clear that C. suffruticosa subsp. algarbiensis is an ornithocoprophil species and its presence on the island was only mentioned in 1989 [19]. Therefore, its abundance in the Berlengas seems to be a direct consequence of the presence of the gull populations, the size of which increased dramatically since the beginning of the twentieth century until 1997, when the colony control measures were implemented [16,48]. Thus, the higher presence of these ruderal species in more disturbed soil, evidenced by PCA results, is not surprising. This is an indicator that the presence of gulls (either nitrification or soil and plant abrasion) enhances the growth of ruderal species, which has been confirmed by other authors. Therefore, we believe that the exclusion zones, along with the absence of mammals, are allowing a slow recovery of the natural rupicolous vegetation on the plateau, where our studies were performed, but the soil is still under the influence of the gull populations, that is, it has been N-enriched, which is leveraging the growth of the nitrophilous vegetation. The seed set found for A. berlengensis was very low when compared to other similar species, such as A. pseudoarmeria (35.2%), A. welwitschii (51.4%) [49] and A. maritima (29.3 seeds per fruit cluster) [50]. These low seed production values for A. berlengensis, Plants 2021, 10, x FOR PEER REVIEW 13 of 19

378 The seed set found for A. berlengensis was very low when compared to other similar 379 species, such as A. pseudoarmeria (35.2%), A. welwitschii (51.4%) [49] and A. maritima (29.3 380 seeds per fruit cluster) [50]. These low seed production values for A. berlengensis, along 381 Plants 2021, 10, 498 with the very high number of unfertilized ovaries (over 50%), indicate that13 of 19pollen disper- 382 sal by insects and birds may be poor, and fertilization is probably scarce. These findings 383 raise a concern about the survival of the species and, along with the known threats already 384 alongstated, with the the low very seed high set number may ofhave unfertilized been a major ovaries issue (over in 50%), the indicate previous that decreases pollen in A. ber- 385 dispersallengensis by populations insects and birdsand the may recruitment be poor, and of fertilization new plants. is probably Despite scarce.the low These number of seeds 386 findingsproduced, raise athe concern dispersal about theand survival germination of the species of the and, few along seeds with produced the known threatsby A. berlengensis 387 alreadyseems stated,to be efficient the low seed enough set may at short have beendistances, a major as issue the inrecruitment the previous data decreases for 2019 in and mainly A. berlengensis populations and the recruitment of new plants. Despite the low number of 388 for 2020 indicate that new plantlets germinate when disturbance decreases. seeds produced, the dispersal and germination of the few seeds produced by A. berlengensis 389 seems toFurther be efficient studies enough will at confirm short distances, if the aspresence the recruitment of the datastructures for 2019 will and allow mainly the plantlets 390 forto 2020grow indicate into adult that newplants plantlets and if germinate these indivi whenduals disturbance will expand decreases. further outside the exclusion 391 areasFurther installed. studies Due will to confirm the slow if the growth presence rate of the of structuresA. berlengensis, will allow these the future plantlets results to will take 392 growa fair into amount adult plants of time and to if acknowledge these individuals extensive will expand recovery further of outside the population. the exclusion areas installed. Due to the slow growth rate of A. berlengensis, these future results will take a fair amount of time to acknowledge extensive recovery of the population. 393 4. Materials and Methods 394 4.4.1. Materials Study Area and Methods 4.1. Study Area 395 The Berlengas Archipelago (39°24′49″ N–9°30′29″ W) is located in the Atlantic Ocean, The Berlengas Archipelago (39◦2404900 N–9◦3002900 W) is located in the Atlantic Ocean, 396 on the Portuguese continental shelf, on the western side of the Iberian Peninsula, close to on the Portuguese continental shelf, on the western side of the Iberian Peninsula, close 397 Cape Carvoeiro (Peniche). It distances approximately 10 km from the mainland and is to Cape Carvoeiro (Peniche). It distances approximately 10 km from the mainland and is 398 composedcomposed of of three three island island groups: groups: Berlenga Berlenga Grande Gr Islandande and Island adjacent and islets adjacent and reefs, islets and reefs, 399 EstelasEstelas Islands Islands and and Farilh Farilhõesões Islands Islands (Figure (Figure9)[1]. 9) [1].

400 Figure 9. Berlengas archipelago location. 401 Figure 9. Berlengas archipelago location. In May 2015, the study area was established in the plateau, where the degradation 402 of graniteIn May rock has2015, allowed the study the accumulation area was established of a thin layer in the of soil. plateau, This is where the preferred the degradation of 403 soilgranite for the rock growth has ofallowedA. berlengensis the accumulation, and indeed, of it wasa thin formerly layer knownof soil. asThis the is “valley the preferred soil 404 offor the theArmeria growth”, where of A. severalberlengensis herbaceous, and speciesindeed, grow, it was as formerly well as the known two varieties as the of “valley of the A. berlengensis, as the name of the valley indicates [51]. Yet, since a survey of 2004 [29], the 405 Armeria”, where several herbaceous species grow, as well as the two varieties of A. ber- number of A. berlengensis was shown to be declining, mainly due to the presence of gull 406 nestslengensis and the, as presence the name of herbivoresof the valley (Figure indicates 10). [51]. Yet, since a survey of 2004 [29], the num- 407 ber of A. berlengensis was shown to be declining, mainly due to the presence of gull nests 408 and the presence of herbivores (Figure 10).

PlantsPlants 20212021, 10, 10, ,x 498 FOR PEER REVIEW 14 of 19 14 of 19

409

410 FigureFigure 10. 10.Location Location of theof the study study area area in Berlengas in Berlengas main island.main island.

411 TheThe area area was was divided divided into 4into subareas, 4 subareas, each one each composed one composed of three squares of three of 10squares× 10 m. of 10 × 10 412 Inm. two In oftwo these of subareas,these subareas, which we which called we “exclusion called areas”,“exclusion one presenting areas”, oneA. berlengensis presenting A. ber- individuals (A1, A2 and A3) and another without any individual of A. berlengensis (B1, B2 413 lengensis individuals (A1, A2 and A3) and another without any individual of A. berlengen- and B3) we installed structuresmade of steel poles and a fishing line, set to prevent the 414 sis (B1, B2 and B3) we installed structuresmade of steel poles and a fishing line, set to approach of gulls (Figure 11a). Each pole was placed along the sides of each square, evenly 415 spacedprevent by the 1 m. approach The fishing of gulls lines were(Figure positioned 11a). Ea parallelch pole to was each placed other, polealong to the pole, sides at of each 416 aboutsquare, 1 m evenly high. In spaced total, 190 by poles1 m. wereThe usedfishing in thelines exclusion were positioned areas. The otherparallel subareas, to each other, 417 againpole oneto pole, exhibiting at aboutA. berlengensis1 m high. growingIn total, (A4,190 poles A5 and were A6) used and another in the exclusion without any areas. The 418 individualsother subareas, of A. berlengensis again one(B4, exhibiting B5 and B6)A. wereberlengensis used as growing controls, and(A4, thus, A5 noand structures A6) and another 419 werewithout placed, any allowing individuals free accessof A. berlengensis by the gulls. (B4, Despite B5 and the B6) setting were of used the structures, as controls, one and thus, 420 gullno structures managed to were set its placed, nest over allowing one of the free largest access individuals by the gulls. of A. berlengensisDespite the, inside setting of the 421 onestructures, of the exclusion one gull areas managed (Figure to11 b).set Yet,its nest thiswas over a uniqueone of event,the largest which individuals has not been of A. ber- recorded on any other occasion, in any other survey. 422 lengensis, inside one of the exclusion areas (Figure 11b). Yet, this was a unique event, Between 2015 and 2020, species presence and cover were assessed in the Spring 423 (15which May, has 16 May,not been 17 May, recorded 18 May, on 19 any May, other 20 Jun) occasion, when most in any species other grow survey. and blossom. Each species cover percentage was registered in a 2 × 2 m square defined in the center of each 10 × 10 m square. During this period, all individuals of A. berlengensis present in the subareas were counted and measured, both in diameter and height, as well as the size structure of the individuals in the exclusion areas. For this purpose, we divided the height of the vegetative plant into 5 categories. Plants smaller than ten centimeters were considered to be plantlets, between 11 and 20 cm young adults, and the other 3 categories were registered as adults (20–30, 30–40 and >41 cm). Additionally, between January and May 2017, 98 fruit clusters (infructescences) from A. berlengensis were harvested. These clusters were collected randomly throughout the main island, from adult plants (height >30 cm) with more than 20 clusters each, 3 clusters being collected from each plant, to prevent significant loss of reproductive units. The seed set was determined based on the total number of seeds per the total number of fruit clusters harvested. Calyxes were also analyzed to determine if ovaries were present and fertilized.

(a) (b)

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409 410 Figure 10. Location of the study area in Berlengas main island.

411 The area was divided into 4 subareas, each one composed of three squares of 10 × 10 412 m. In two of these subareas, which we called “exclusion areas”, one presenting A. ber- 413 lengensis individuals (A1, A2 and A3) and another without any individual of A. berlengen- 414 sis (B1, B2 and B3) we installed structuresmade of steel poles and a fishing line, set to 415 prevent the approach of gulls (Figure 11a). Each pole was placed along the sides of each 416 square, evenly spaced by 1 m. The fishing lines were positioned parallel to each other, 417 pole to pole, at about 1 m high. In total, 190 poles were used in the exclusion areas. The 418 other subareas, again one exhibiting A. berlengensis growing (A4, A5 and A6) and another 419 without any individuals of A. berlengensis (B4, B5 and B6) were used as controls, and thus, 420 no structures were placed, allowing free access by the gulls. Despite the setting of the 421 Plants 2021, 10, 498 structures, one gull managed to set its nest over one of the largest individuals of A.15 ofber- 19 422 lengensis, inside one of the exclusion areas (Figure 11b). Yet, this was a unique event, 423 which has not been recorded on any other occasion, in any other survey.

(a) (b)

Figure 11. (a) Study areas deﬁned in the plateau of Berlengas Island: “structure + Armeria” A(1–3): exclusion area with A. berlengensis growing; “no structure + Armeria” A(4–6): no structures installed with A. berlengensis growing; “structure + no Armeria” B(1–3) exclusion area without any A. berlengensis; “no structure + no Armeria” B(4–6): no structures installed in an area without any A. berlengensis.(b) Nest of a Yellow-legged Gull on an adult A. berlengensis, summer 2017, exclusion zone A3. The partial destruction of the plant is easily seen, both due to gull-tramping and to nitrophilous excrements.

4.2. Armeria berlengensis Morphology and Distribution A. berlengensis is a sturdy bushy plant, up to 80 cm (130 cm) in diameter, and with aerial branches of variable lengths (up to 50 cm). Leaves 30–70 × 3–14 mm are arranged in terminal tufts, from linear-lanceolate to lanceolate, usually with 3 to 5 nerves, ﬂat and fairly rigid. External bracts are oval-shaped, cusped, longer than those of the middle part, the internal ones are obovate-oblong, mucronate. The calyx is 6–9 mm, with rows of nurtured hairs, with these being more than 0.3 mm long. Corolla is pink to white [9]. Two varieties of the species have been acknowledged on the archipelago, var. berlengensis which has glabrous leaves and var. villosa with villous leaves [51–53]. Pollen is transported by nonspecialized insects and, less frequently, by birds [30,54]. Like all the other Armeria species, A. berlengensis seeds are adapted for wind dispersal and are dispersed enclosed in the calyx, as suggested by the parachute-like shape of the calyx [50]. The more extreme habitats are represented by the refuge of native stress-tolerant vegetation, such as A. berlengensis [9,16,29,42]. A. berlengensis occurs throughout the archipelago, mainly on rocky outcrops, halophytic cliffs and consolidated gravel pits under the inﬂuence of wave spray and strong winds (Figure 12). Plants 2021, 10, 498 16 of 19 Plants 2021, 10, x FOR PEER REVIEW 16 of 19

462 463 FigureFigure 12.12. DistributionDistribution ofof ArmeriaArmeria berlengensis on Berlengas island, island, da datata collected collected from from 2016–2018. 2016–2018. 464 AdaptedAdapted from from [ 16[16].].

465 4.3.4.3. StatisticalStatistical AnalysisAnalysis 466 ToTo study study the the number number of ofA. A. berlengensis berlengensisindividuals individuals in in the the exclusion exclusion areas areas A(1–3) A(1–3) and and 467 thethe controlcontrol areasareas A(4–6),A(4–6), thethe parametricparametric Student’sStudent’s t-testt-test waswas performedperformed [[55],55], globally,globally, as as 468 wellwell as as within within each each sampling sampling period period (Spring (Spring 2015 2015 to Springto Spring 2020). 2020). Additionally, Additionally, to compare to com- 469 thepare cover the percentagecover percentage across across the study the periodstudy (2015–2020)period (2015–2020) of A. berlengensis of A. berlengensisin the Spring, in the 470 anSpring, analysis an analysis of variance of variance (ANOVA) (ANOVA) with one wi factorth one wasfactor performed. was performed. The procedure The procedure was 471 performedwas performed globally, globally, as well as as well for eachas for type each of type structure of structure (namely, (namely, exclusion exclusion areas A(1–3) areas 472 “structureA(1–3) “structure + Armeria + ”Armeria and the” and control the control areas A(4–6) areas A(4–6) “No structure “No structure + Armeria + Armeria”). All”). data All 473 weredata checkedwere checked for normality for normality and homoscedasticity and homoscedasticity [55]. Whenever [55]. Whenever the assumptions the assumptions were 474 notwere met, not the met, Kruskal–Wallis the Kruskal–Wallis non-parametric non-parame testtricwas test performed.was performed. Whenever Whenever applicable, applica- 475 Tukeyble, Tukey or Dunn or Dunn multiple multiple comparison comparison tests tests were were used used (according (according to to the the ANOVA ANOVA or or the the 476 Kruskal–WallisKruskal–Wallis test, test, respectively). respectively). Additionally, Additionally, to to evaluate evaluate the the strength strength of of the the correlation correlation 477 betweenbetween the the mean mean height height and and the the mean mean diameter, diameter, across across the the study study period period (2015–2020) (2015–2020) of A. of 478 berlengensisA. berlengensisin the in Spring,the Spring, Pearson’s Pearson’s correlation correlation coefficient coefficient (r) was (r) used was [us55ed]. Results [55]. Results were 479 consideredwere considered significant significant at p-value at p-value≤ 0.05 ≤ level.0.05 level. Where Where applicable, applicable, results results are presentedare presented as ± ± 480 meanas meanstandard ± standarddeviation deviation (SD) (SD) or or mean mean ±95% 95% confidence confidence interval interval (CI). (CI). All All calculations calculations 481 werewere mademade withwith IBMIBM SPSS SPSS Statistics Statistics version version 27 27 software. software. Finally,Finally, principal principal components components analysis (PCA), based on correlation matrices, was applied to identify the main associations 482 analysis (PCA), based on correlation matrices, was applied to identify the main associa- among the cover of the observed species and the areas (with A. berlengensis exclusion area 483 tions among the cover of the observed species and the areas (with A. berlengensis exclusion and control, without A. berlengensis exclusion area and control) and sampling periods 484 area and control, without A. berlengensis exclusion area and control) and sampling periods (Spring 2015 to Spring 2020). The principal component analysis (PC1 and PC2) provides 485 (Spring 2015 to Spring 2020). The principal component analysis (PC1 and PC2) provides information on the most meaningful parameters, which describe a whole data set, affording 486 information on the most meaningful parameters, which describe a whole data set, afford- data reduction with minimum loss of original information. Although the results concerning 487 ing data reduction with minimum loss of original information. Although the results con- the first two components were presented, the others were also analyzed. To perform the 488 cerning the first two components were presented, the others were also analyzed. To per- analysis, only those species with an annual percentage of coverage higher than 2% were 489 form the analysis, only those species with an annual percentage of coverage higher than considered, with the remaining species being considered as a record without significant 490 2% were considered, with the remaining species being considered as a record without expression. Values expressed as a relative percentage were arc-sine square-root transformed 491 significant expression. Values expressed as a relative percentage were arc-sine square-root before analysis [56]. All calculations were performed with the CANOCO version 4.5 492 transformed before analysis [56]. All calculations were performed with the CANOCO ver- package [57]. 493 sion 4.5 package [57]. 494

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5. Conclusions The survival of the natural rupicolous vegetation of Berlenga Island faces several challenges due to the disturbance the island has been subjected to in recent decades. PCA showed two distinct clusters of species, namely, natural rupicolous species and nitrophilous vegetation, showing the major influence that herbivores and gulls have had on the island’s vegetation. The installation of protective structures (exclusion zones) that prevent the access of gulls, along with the removal of rats and rabbits, seem to have played an important role on the recovery of the natural rupicolous vegetation, namely on A. berlengensis. The number of seedlings increased and significant differences in height and diameter were noticeable, mainly for 2020. However, in addition to its slow growth, the particularly low seed set of A. berlengensis hinders the rapid recovery of the species. Thus, a longer experimental period is necessary to corroborate if seedling establishment is successful and, thus, if these conservation measures are truly effective. Although this is a preliminary study, some considerations can be already made regarding the population of A. berlengensis. The presence of man, gulls, and C. edulis in other locations of the main island, is enriching the soil nitrate and phosphate contents, which favors the growth of ruderal vegetation at the expense of rupicolous vegetation [58]. Therefore, the control of these problematic issues seems to be the best way to allow the recovery of native vegetation. On the one hand, controlling the touristic activities, human construction and trampling on the island, is of utmost importance. These measures have already been implemented in the management plan of the Berlenga Reserve, in particular, with the implementation of a carrying capacity on the island. On the other hand, continued control of the gull population, and the installation of other exclusion areas will reduce soil nitrification and gull trampling, although much of the plateau’s soil has already been degraded, hindering the growth of rupicolous species. Finally, the complete removal of mammals will favor the growth of shrubs instead of annual vegetation, and the control of C. edulis will allow a recovery, even if slow, of native vegetation. Further studies of the exclusion areas and outside, of the rupicolous and the nitrophilous vegetation on the next years will provide us with more clues into the main factors influencing the already visible trend of the Berlenga vegetation.

Author Contributions: Conceptualization, T.M., S.M., and C.A.; methodology, T.M., S.M., and C.A.; validation, T.M., S.M., L.M., A.I.F., and C.A.; formal analysis, T.M., S.M., and C.A.; investigation, T.M., S.M., I.F., A.I.F., L.M., N.O., P.C., and C.A.; data curation, T.M., S.M., I.F., A.I.F., L.M., N.O., P.C., and C.A.; writing—original draft preparation, T.M..; writing—review and editing, T.M., A.I.F., N.O., L.M., S.M. and C.A.; supervision, T.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by LIFE + Program LIFE13 NAT/PT/000458 LIFE Berlenga and Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2020 granted to MARE. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The research has been conducted in compliance with the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Specimens of A. berlengensis are deposited in the Herbarium of the University of Coimbra, Portugal (COI00084706, COI00084707, COI00084687). Acknowledgments: To the long list of volunteers who helped in the fieldwork during the project coordinated by SPEA and to the other remaining staff of the ICNF—Instituto da Conservação da Natureza e das Florestas, Portugal, special thanks are due for all logistic support and on the plants’ collection from the Natural Reserve of Berlenga Island. An acknowledgement is also due to the Portuguese Navy and the company Berlenga Praia-Actividades Marítimo Turísticas, Lda., owner of the boat “Julius” for the transportation provided to and from Berlenga Island. Conflicts of Interest: The authors declare no conflict of interest. Plants 2021, 10, 498 18 of 19

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