Hybridization processes in an introduced subpopulation of an endangered plant: Management strategies to guarantee the conservation of Helosciadium bermejoi (Apiaceae)

Accepted Manuscript Title: Hybridization processes in an introduced subpopulation of an endangered plant: Management strategies to guarantee the conservation of Helosciadium bermejoi (Apiaceae) Authors: Juan Rita, Miquel Cap´o, Eva Moragues, Josefina Bota, Joana Cursach PII: DOI: Reference:

S1617-1381(17)30236-4 https://doi.org/10.1016/j.jnc.2017.10.006 JNC 25590

To appear in: Received date: Revised date: Accepted date:

12-5-2017 15-9-2017 24-10-2017

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Hybridization processes in an introduced subpopulation of an endangered plant: management strategies to guarantee the conservation of Helosciadium bermejoi (Apiaceae) Juan Rita (1), Miquel Capó(1), Eva Moragues(2), Josefina Bota (1) and Joana Cursach(1) (1)

Grup de Recerca en Biologia de les Plantes en Condicions Mediterrànies, Dep. de Biologia,

Universitat de les Illes Balears. Cra. Valldemossa km 7,5. E-07122 Palma. Spain (2)

Direcció General d'Espais Naturals i Biodiversitat, Conselleria de Medi Ambient, Agricultura i Pesca,

Govern de les Illes Balears. C/ Gremi Corredors 10, Polígon Son Rossinyol, E-07009 Palma, Spain Corresponding author: Juan Rita, [email protected], +34971173180, ORCID ID 0000-0002-9372-1637

Abstract Translocations are a tool to restore populations of threatened plant species that are being widely used. However, these techniques are not without risks and require rigorous protocols to carry them out and for their subsequent monitoring. In translocation projects, hybridization with other species is one of the most important risks and may threaten the survival of the species we want to protect. Here we present the case of Helosciadium bermejoi (L. Llorens) Popper and M.F. Watson, a Critically Endangered plant from the island of Menorca (western Mediterranean). It occurs only in one locality and has been introduced to three new locations during recent years according to its Recovery Plan (2008). Recently, intermediate forms between the endangered species and a native congener, Helosciadium nodiflorum (L.) Koch, have been detected. The aims of this study are (i) to elucidate whether these plants originated through a hybridization event and (ii) to provide information about the current conservation status of the species. Exhaustive monitoring of all subpopulations (number of patches and area of occupancy) was performed from September 2015 to June 2016, and the rDNA ITS region and cpDNA rps16-trnK intergenic spacer region were sequenced for several samples of the putative hybrid and its putative parental species. Overall, the entire population of H. bermejoi greatly increased from 2010 (last census with available data) to 2015 in terms of both number of patches and area of occupancy: from 110 to 277 patches and from 299.2 to 791.3 dm2. Molecular analysis confirmed the hybrid origin of Helosciadium × clandestinum Rita, Capó and Cursach and that both H. bermejoi and H. nodiflorum (L.) Koch can act as donors of

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pollen or ovules. We discuss the advantages and disadvantages of translocations, as well as the great importance of monitoring them for at least the next ten years, both to know if the actions have been successful and to prevent or mitigate risks. We also present this case as an example for the importance of fast decision and coordination between environmental managers. Key words: Conservation biology, Balearic Islands, Hybrid management, Apium, Assisted colonization, Mediterranean flora, Endemic plant

Introduction Islands and archipelagos have enormous value in terms of plant biodiversity, where high level of endemicity are common due to remarkable processes of speciation and adaptive radiation (Kier et al. 2009; Caujapé-Castells et al. 2010; Whittaker et al. 2017). In addition, they may have also acted as climate refuges, conserving species that had a wider distribution in the past (Médail and Diadema 2009). However, they are also places where threats are very important and where plant extinction events have been particularly notable, close to 75% of plant species extinctions of the last 500 years have happen in islands (Sax and Gaines 2008; Caujapé-Castells et al. 2010). Because of its vulnerability many islands and archipelagos have been included as biodiversity hot spots (Médail and Quézel 1997). Management techniques of threatened plant species have notably been developed in recent years (Bañares 2003; Marrero et al. 2015; Goñi et al. 2015). In-situ and ex-situ actions are needed to decrease the probability of species extinction, especially in extremely narrow endemic plants. Translocation is frequently used to guarantee the demographic and genetic restoration of threatened species (Akeroyd and Jackson 1995; Guerrant and Kaye 2007; Menges 2008; Kramer and Havens, 2009; Weeks et al. 2011; Ren et al. 2012), even in assisted migrations, when habitat has been lost due to climate change (McLachlan et al. 2006; Kramer and Havens 2009; Vitt et al. 2010; Gómez et al. 2015). However, some authors warn of the need for having enough information before acting and to carry out a risk analysis (Hewitt et al. 2011). Moreover, its viability has been questioned because even if preceded by careful risk assessment, such action is likely to produce unintended and unpredictable consequences (Ricciardi and Simberloff 2009). The International Union for Conservation of Nature (IUCN/SSC 2013) states that conservation translocation is the intentional movement and release of a living organism where the primary objective is a conservation benefit; this will usually comprise improving the conservation status of the focal species

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locally or globally and/or restoring natural ecosystem functions or processes. These translocations can act to restore the original population either by reinforcing existing populations or through reintroductions inside the indigenous range from which it has disappeared. On the other hand, the term benign introduction is also widely used and consist of the intentional movement and release of an organism outside its indigenous range to avoid the extinction of populations of the focal species (Escudero and Iriondo 2003).

Translocations also have risks; large quantities of resources and time are needed, and success is often difficult to guarantee (Maunder 1992; Godefroid et al. 2011; Weeks et al. 2011; IUCN/SSC 2013; Fenu et al. 2016). In fact, these actions should only be chosen after an extensive risk and viability analysis (Escudero and Iriondo 2003). Some authors recommended that they should be considered only as the last resort when habitat protection is not possible or the taxon is critically endangered to become extinct and appropriate sites and propagule source materials are available (Escudero and Iriondo 2003; Maschinski et al. 2012). Hybridization with related taxa is one of the most important risks of translocation (IUCN/SSC 2013). These events can cause outbreeding depression, demographic swamping or genetic assimilation (Levin et al. 1996; Levin 2002; Arrigo et al. 2016). The survival of rare or threatened species has been affected by hybridization in numerous occasions, most of which were caused by environmental perturbations where congeners were brought into artificial sympatry through human actions, including agricultural activities, land use changes, and the spread of invasive species (Levin et al. 1996; Lamont et al. 2003; Fant et al. 2010; Weeks et al. 2011; van Hengstum et al. 2012; Piett et al. 2015). Hybridization is especially deleterious when it occurs in narrow endemic species; if it presents weak prezygotic isolation, hybridization can cause extinction events (Levin et al. 1996; Weeks et al. 2011; IUCN/SSC 2013). In insular areas, the extinction risk increases due to the presence of weaker genetic isolation barriers compared to continental congeners (Levin et al. 1996). Hybridization events can be detected using molecular techniques, especially in F1 offspring. The analysis of the internal transcribed spacer (ITS) is considered a useful resource for phylogenetic works (Baldwin 1992; Baldwin et al. 1995) and has been widely employed in studies of plant hybridization (Hegarty and Hiscock 2004; Conesa 2010). On the other hand, cpDNA allows the detection of maternity and paternity roles because it is maternally inherited in almost all angiosperms (Ennos et al. 1999). In this sense, the rps16-trnK intergenic spacer region of cpDNA has been used in key studies of phylogeny in the Apiaceae family (Lee and Downie 2006; Spalik et al. 2009) to establish maternity (Desjardins et al. 2015).

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Helosciadium bermejoi (L. Llorens) Popper and M.F. Watson (Ronse et al. 2010) (= Apium bermejoi L. Llorens 1982) is a narrow endemic plant from Menorca island (Balearic Islands, Spain). It is a small plant which live in a single locality, in a short seasonal torrent near the coast. This site has been protected as a plant Micro-Reserve. The total number of mature patches of the original population did not exceed one hundred (Rita and Cursach 2013). The reasons of their narrow distribution are unknown, but reduction of suitable natural habitat and competition with native species are the main hypothesis proposed (Rita & Cursach 2013). Given the small size of the only natural population of H. bermejoi, and according to the IUCN (2012) criteria its extinction risk was very high. So, following a Recovery Plan of this species the environmental authority decided, among other actions, to create a seed bank, to cultivate the plant in several botanical gardens and to introduce the species into new sites to reduce the probability of extinction (Rita and Cursach 2013). During the spring of 2015, in one new locality where a translocation actions had been done (2008), unidentified individuals among the H. bermejoi subpopulation were found. These plants had an intermediate morphology between H. bermejoi and Helosciadium nodiflorum (L.) Kock, and they were considered to be a new hybrid, which was formally described as Helosciadium × clandestinum Rita, Capó and Cursach (Rita et al. 2016). Because hybridization would be a serious threat to the survival of H. bermejoi, its detection was also considered very relevant for the conservation and management of the focal species. The aims of this study were to (i) monitor both the natural and introduced subpopulations to assess the conservation status of H. bermejoi in the wild, (ii) determine by molecular analysis whether the intermediate forms between H. bermejoi and H. nodiflorum found in one translocation locality originated by a hybridization event, and (iii) discuss the risk of translocations and the complexity of decision making in the management of hybrid populations.

Methods Study system H. bermejoi is an endangered plant endemic to Menorca Island (Balearic islands, Western Mediterranean basin), it is a small (1−4 cm high) stoloniferous plant that inhabits one small locality on the north-eastern coast of Menorca, divided into two subpopulations (CNe and CNe2). It inhabits the moist bed of siliceous torrents (Paleozoic turbidites) in an area with strong influences from the sea and wind. The

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impermeability of the substrate causes some areas to remain flooded with running water during the rainy season (from September to April) but to dry out completely during the rest of the year. A detailed description of the species and its reproductive biology can be found in Llorens (1982), Knees (2003) and Cursach and Rita (2012). It is considered Critically Endangered according to the criteria of the IUCN (2012) (Montmollin and Strahm 2005; Moreno 2008). To reduce the probability of extinction first, a pilot translocation project (CNo) was conducted in 2005 following the criteria of a prior introduction plan (Rita and Cardona 2004). Afterwards, in 2008, the Govern de les Illes Balears approved a “Recovery Plan of Apium bermejoi” (Resolution 8245 of 2008). During the execution of this Recovery Plan, the species was successfully introduced into two new locations (PF and MV), and the total population and localities inhabited by this species increased notably (Rita and Cursach 2013). All of the new localities are located in the protected area of the Natural Parc of S’Albufera des Grau in the Northeast coast of Menorca, and the chosen sites had the same substrate and similar ecological characteristics than the original (for more geographical details see Rita and Cursach 2013). H. bermejoi is close related to other species of the same genus, such as Helosciadium nodiflorum (L.) Koch (=Apium nodiflorum (L.) Lag.) and Helosciadium repens (Jacq.) Koch (=Apium repens (Jacq.) Lag. (Spalik et al. 2009; Ronse et al. 2010). Only the first one occurs in Menorca (Fraga et al. 2004) but there are not any population almost 5 km around the original population of H. bermejoi. H. nodiflorum can hybridize with H. repens (Apium × longipedunculatum (F.W. Schultz) Rothm. (Grassly et al. 1996, Desjardins 2015), with Helosciadium inundatum (L.) Koch (Helosciadium × moorei (Syme) Bab. = Apium x moorei (Syme) Druce.), and with Berula erecta (× Beruladium procurrens A.C. Leslie) (Desjardins et al. 2015).

H. nodiflorum has rooting stem nodes, like H. bermejoi, but it is much bigger, it grows more than 100 cm high, usually remains erect instead of lying on the substrate and tends to live in water-covered areas. Nevertheless, this species presents huge variability in its size and shape, and it may also appear in plastodemes of very small plants that colonize open wet areas (plastodeme: assemblages of plants phenotypically rather than genetically distinct) (McDonald and Lambrick 2006). It is a broadly distributed North Hemispheric species, usually lives in torrents, wetlands and ponds, in which it behaves as a helophyte. However, the species is able to colonize wet soils that are not flooded at the margins of those

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habitats, in which cases it decreases dramatically in size and adopts a prostrated shape with umbels close to the soil surface (plastodemes). See McDonald and Lambrick (2006) for more details about its biology.

Monitoring of natural and introduced subpopulations of Helosciadium bermejoi Both the original (CNe and CNe2) and introduced subpopulations (CNo, PF and MV) were monitored in mid-June and September of 2015 and January and June of 2016. This plant forms dense lawns of leaves and stems so that its seeds tend to germinate very close together, and new plants grow to form clusters in which individuals very quickly become indistinguishable. Based on a previous work (Rita and Cursach 2013), we have chosen to monitor a "patch" of H. bermejoi as a sample unit, which could consist of one or more individuals. Each patch was identified according to its position in a two-axis system following the methodology in Rita and Cursach (2013). The length and width of all patches were measured to estimate their area of occupancy. In the cases of patches with an irregular form, we applied estimates of the % cover as a correction factor. In cases where most of the patches had dry cover (CNe subpopulation in June 2015; CNe, CNe2 and PF subpopulations in June 2016), we also applied estimates of the % of green cover.

Molecular analysis of hybrid individuals Plant material of H. bermejoi, H. nodiflorum and H. × clandestinum were obtained from different locations in fresh or silica gel-dried status as described in Table 1. Prior to the molecular analysis, vouchers of all samples were made and deposited in the herbarium of the Universitat de les Illes Balears (UIB). Total DNA was extracted from 100 mg of leaf tissue using the cetyl trimethylammonium bromide (CTAB) method (Doyle and Doyle 1987), adding 3% (w/v) polyvinylpyrrolidone (PVP) during the grinding step to avoid the presence of polyphenolic compounds (Porebski et al. 1997; Sharma et al. 2002). ITS and rps16-trnK intergenic spacers were amplified by polymerase chain reaction (PCR). The reactions were conducted in a total volume of 20 µL containing 800 ng DNA template, 250 µM of each dNTP, 0.5 µM of each primer, 1 U of Ex Taq Polymerase and 1x Ex Taq Buffer (Takara Ex Taq; Takara, Kyoto, Japan). The ITS region was amplified with the primers ITS-1 (5’ TCC GTA GGT GAA CCT GCG G 3’) and ITS-4 (5’ TCC TCC GCT TAT TGA TAT GC 3’) (White et al. 1990) under the cycling conditions 94°C/03:00 + 40 × (94°C/00:30, 57°C/00:30, 72°C/01:00) + 72°C/5:00, and the rps16-trnK 6

intergenic spacer was amplified using the primers 3’ exon (5’ TTC CTT GAA AAG GGC GCT CA 3’) and trnK (5’ TAC TCT ACC GTT GAG TTA GC 3’) (Lee and Downie 2006) under the cycling conditions 94°C/01:00 + 30 × (94°C/00:30, 55°C/01:00, 72°C/01:00) + 72°C/02:00 using an Eppendorf Mastercycler (Eppendorf, Westbury, NY). PCR products were separated in 3% agarose gels and were run at 120 V for 40 min to verify their quality. Amplicons were purified by the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals) according to the manufacturer’s instructions and were sequenced directly. To determine the hybridization events and hybrid maternal inherence, sequences were generated for all samples and phylogenetic trees were also obtained (Desjardins et al. 2015). Sequence reads were trimmed, adjusted and blasted using the BioEdit sequence alignment editor (Hall 1999). The ITS sequences of H. bermejoi and H. nodiflorum were compared with related accessions in the GenBank database (Supplementary Information 1) in order to find species-specific markers and verify ambiguity in the sequences of putative hybrids. Maternal inherence (Ennos et al. 1999) was detected by blasting the rps16-trnK sequences of putative hybrids against both species. Maximum parsimony analysis (MP) was executed in PAUP* 4.0 (Swofford 2002) using a branch and bound search and the furthest method for the topology investigation following the procedures of Desjardins (2015). Bootstrap values were calculated for 1000 replicates using the MP heuristic search strategy (Felsenstein 1985) to estimate node support for the inferred trees.

Results Monitoring of natural and introduced subpopulations of Helosciadium bermejoi Subpopulations monitoring was analysed using two variables: the number of patches and the estimation of the area of occupancy for each subpopulation (Table 2). Because the seeds tend to germinate very close to one another, several individuals can eventually appear to be mixed in a single patch during the spring when they obtain their maximum size. So the values of the coverage provide more real idea of the state of the population during the spring than the number of patches, in contrast the number of individuals is more significant in the winter censuses. In the original locality (CNe), the number of patches in June increased in relation to the last period for which data is available (from 55 in June 2010 to 95 in June 2016). However, the (green) area of occupancy decreased (from 131.56 dm2 in June 2010 to 71.87 dm2 in June 2016) (Figure 1). The CNe2 7

subpopulation, which is located a few hundred meters from the other, consisted of three isolated patches with a total cover area of 12.81 dm2 in June 2016, representing only 5% of the coverage at CNe (similar values to those recorded in June 2010, Rita and Cursach 2013). When the original locality (CNe) was monitored in June, the torrent bed was dry both in 2015 and 2016; therefore, the total area of occupancy (i.e., including the dry cover) represented the maximum vegetative development for each season (see Cursach and Rita (2012) for phenology details). According to this value, the original locality showed the highest area of occupancy in 2016 of all the years for which data are available (Figure 2). The introduced subpopulation at CNo showed just three patches with a cover of 4.63 dm2, similar values to those in June 2010 (4 patches with a cover of 9.75 dm2, excluding plants planted in the reinforcement in May 2010, Rita and Cursach 2013). In PF and MV, subpopulations introduced in 2008, the areas of occupancy in June 2015 were 362.07 dm2 and 192.46 dm2, respectively (Table 1). These values were considerably higher than those observed in June 2010 (Figure 2). Regarding the number of patches, in PF, this value increased from 28 to 171; however, in MV, the numbers of patches were similar (21 and 23, respectively). In both localities, H. bermejoi has colonized areas at distances of several meters from the points where plants were planted in the introduction project (personal observations). Overall, the whole population of H. bermejoi has greatly increased from 2010 to 2015 in terms of both number of patches and area of occupancy: from 110 to 277 patches and from 299.18 to 791.32 dm2. The rise in these values was especially attributed to the PF and MV subpopulations. In the census of June 2015, the MV subpopulation contributed up to 30.26% to the green area of occupancy of the whole population of this endangered species.

Helosciadium × clandestinum in Mongofre Vell, actions for conservation In the census of June 2015, some very vigorous plants morphologically differentiated from H. bermejoi (e.g., presence of compound umbels) and H. nodiflorum (e.g., umbels with less than 4 rays) were detected in the introduced MV subpopulation. These characters were compatible with hybridization between these two species (this hybrid was formally described as H. × clandestinum Rita et al. (2016)). At that time, the putative hybrid and putative parental species were in full bloom but with unripe fruits. This information was immediately transferred to the environmental authority (Conselleria de Medi Ambient, Agricultura i Pesca del Govern Balear, CMA). In common agreement, it was found that a) a vigorous hybrid was a

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serious threat to the conservation of H. bermejoi, even without a formal recognition; b) this hybrid had recently arisen due to the fact it was not detected in the previous censuses; c) the fruits produced that year from both H. bermejoi and H. nodiflorum in the area were genetically dubious; d) fructification was still at an early stage, so these fruits had not yet dispersed; and e) the risk of hybridization will continue occurring at that locality in the future. From these assumptions, it was necessary to prevent the putative hybrid from expanding into the natural environment and ensure that it could not appear again in the future. To achieve both goals jointly, it was decided to perform the following actions: a) remove all hybrid individuals using strict control to ensure that no propagule could disperse into the environment (plants and associated topsoil were extracted from the site, plant material was dried and preserved in the herbarium of the UIB, and the soil was sterilized in an oven and discarded; b) eradicate all individuals of this introduced population of H. bermejoi in addition to the associated topsoil; c) remove all individuals of H. nodiflorum in the same area and the umbels of this species in the perimeter (these actions were performed by members of the UIB, CMA and Consell Insular de Menorca (CIMe) six days after the location of hybrids); and d) monitor subpopulations in the subsequent months and years to remove individuals of any of the three entities that may emerge. In this way, plants of H. × clandestinum were also found in September of 2015 (one plant) and in January (one plant), May (one plant) and June of 2016 (two plants). Both hybrid plants found in June of 2016 were in reproductive status. These hybrids plants were also removed from the field and were used for molecular studies and maintained in the experimental field of the UIB. New plants found of H. bermejoi and H. nodiflorum were removed as well.

Molecular analysis DNA sequencing of the rDNA ITS region and the cpDNA rps16-trnK intergenic spacer region was implemented using fifteen samples: six of H. bermejoi (four from MV and two from CNe; both located in Menorca), five of H. nodiflorum (three from Menorca, sampled in three separated locations, and two from Estellencs, Mallorca) and four of the putative hybrid from MV. The generated ITS sequences were approximately 580 bp in length, and the GC average was approximately 55.69%. The GenBank accessions of H. bermejoi and H. nodiflorum showed six speciesspecific markers in the positions 12, 69, 89, 109, 446 and 472 as described in Table 3. Because position 109 was unequal between the H. nodiflorum accessions and the two sampling locations of this study, this position was not considered species-specific and was discarded for hybrid identification. Moreover, in the 9

H. nodiflorum sequences of this study, an insertion of an adenine was detected in position 396, so comparisons of positions 446 and 472 could not be conducted because of the ambiguous reads in the hybrid sequences. To verify how much the difference at position 109 and the insertion at position 396 can affect the H. nodiflorum characterization of paternal samples, a phylogenetic analysis of the Helosciadium genus and related species of the Apiaceae family was performed, and a gene tree based on ITS sequences is represented in Figure 3. The four populations of H. nodiflorum used in this study (MVN, BG, TOR & ES) were placed in the H. nodiflorum cluster (95% bootstrap support). Electrophoretograms of the four putative hybrid sequences showed ambiguous curves in (and only in) the four informative positions (Supplementary Information 2). Moreover, two samples of H. bermejoi samples taken one year later (MV-F1) showed no effects of natural retrohybridization. The rps16-trnK intergenic spacer generated sequences that were approximately 830 bp length with an average GC content of 25.8% and found six informative markers of inherence. All the samples were directly blasted with samples of parental species from the same location and showed 100% similarity with one of the parents, as is represented in Table 3. A phylogenetic tree based upon maximum parsimony analysis of rps16-trnK intergenic spacer sequence data assigned the putative hybrids to separate clades, one sample was placed in the H. bermejoi clade (100% BS), while the other three samples were placed in the H. nodiflorum clade (100% BS), as shown in Figure 4.

Discussion Assessment of introduction performance The introduction of threatened species into new localities outside their known range is a controversial conservation action, and, in any case, it should be the last option even in critical situations (Escudero and Iriondo 2003; Ricciardi and Simberloff 2009; Weeks et al. 2011; Maschinski et al. 2012; IUCN/SSC 2013). It is also complicated to determine when an introduction has been successful (Guerrant and Kaye 2007; Rossi and Bonomi 2007; Menges 2008; Godefroid et al. 2011). H. bermejoi, with a single natural site and a highly fluctuating population of less than a hundred breeding patches, met the requirements for undertaking benign introductions to reduce both environmental and demographic stochastic risks (Rita and Cursach 2013). The benign introductions carried out in 2008 at two new locations, in addition to a

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third one previously carried out in 2005, have been reviewed eight years later. In the spring of 2015, the introduced subpopulations accounted for 70.6% of the total area of occupancy of the species in the wild. This occurred at a time when the original subpopulation reached its historical maximum. Therefore, the overall cover of the species in the wild in 2015 was more than twice as high as in 2010, at the time of the last assessment by the authors. The majority of the individuals of all the subpopulations were reproductive, and the germination and establishment of new seedlings were verified in all of them. These data confirm the conclusions of Rita and Cursach (2013), in that these benign introductions were successful, since they meet the most widely accepted criteria regarding the evaluation of introductions (Guerrant and Kaye 2007; Rossi and Bonomi 2007; Menges 2008; Godefroid et al. 2011). Two of the introduced subpopulations (MV and PF) were the most important in terms of both the number of patches and area of occupancy. Both cases show that benign introductions constitute a viable method to significantly increase the population of this species and reduce the risks associated with a very small population living at a single locality. This success was due to the correct selection of suitable localities (similar habitats than the original population) and the large number of individuals (about one hundred in each locality) that were planted. This allowed the individuals to be distributed in different microhabitats (substrates with more or less humid conditions) to guarantee that some of them would be optimal for the species (for more details about the criteria to chose the new site, selection of plant material and methods of sowing see Rita & Cursach (2013)). In the following years, natural fruit dispersion facilitated the colonization of the best microhabitats, while the species disappeared from those not suitable enough. It is worth highlighting the increase in the cover area for those subpopulations from 2010 to 2015 (in MV and PF, the area of occupancy quadrupled and tripled, respectively). The third introduced subpopulation (CNo) remained stable over the years but always with a low number of individuals. This subpopulation is interesting because it is an indicator of the resistance of this species from disappearing even when the number of individuals in the subpopulation is very low. Indeed, the same behavior has been observed in the natural subpopulation of CNe2, which has remained stable after more than fifteen years of monitoring, despite having a number of patches below 10. This resistance is surprising for a plant with strong annual demographic fluctuations and that quickly disappears when the conditions are not adequate. On other hand, it shows how the bottleneck that led to total genetic uniformity of cpDNA (Rosselló 2004, report unpublished (LIFE2000NAT/E/7355)) could have occurred in the past. These very small subpopulations are maintained over time by replacing individuals with new

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seedlings rather than by the persistence of mature individuals (Rita and Cursach 2013). In this sense, their persistence depends on the production of new seeds and the survival of the seedlings each year. Unfortunately, the trends of climate change in the Balearic Islands show an expansion of summer and a reduction of spring and possibly autumn (Jansa et al. 2017). This scenario increases the vulnerability of H. bermejoi, as it directly affects its flowering and fruiting (May-June) and seed germination (mainly in September-October), making it more likely that a particularly hot and dry year will cause the failure of fruiting and/or germination.

A new human-induced hybrid plant The sequences of the rDNA ITS region of all H. × clandestinum individuals analysed shows ambiguity in the four species-specific markers, which are those that are different between H. bermejoi and H. nodiflorum. This is an objective confirmation of its hybrid origin. Despite differences found in the ITS region between Balearic and continental populations of H. nodiflorum, comparing sequences of H. × clandestinum and H. nodiflorum plants from the Menorca populations can confirm the parental link for H. x clandestinum. On the other hand, the rps16-trnK intergenic spacer region from the cpDNA of H. × clandestinum individuals collected in the field showed that both H. bermejoi and H. nodiflorum can act as donors of pollen or ovules and that hybridization can therefore occur in both directions. These data suggest that hybrids could have originated among either H. bermejoi or H. nodiflorum patches. However, after a thorough exploration of the torrent outside the area occupied by H. bermejoi but with H. nodiflorum, no individual was found with the characteristics of the hybrid. Some doubtful individuals found in the areas where only H. nodiflorum was present were also analysed, and their ITS region sequences allowed us to identify them as pure H. nodiflorum (data not shown). Therefore, the most likely scenario is that hybridization occurred only in places where the two species make physical contact and that this hybridization event was very recent and likely in the F1 stage and that preferential pollination by ants should limit pollen transport over intermediate and long distances. However, this risk is not zero because visits by flying insects have also been observed (Cursach and Rita 2012). Consequently, the risk of the creation of new hybrids in the other introduced subpopulations is small but not negligible, so periodic monitoring is essential to avoid any risk.

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This hybridization occurred after an extraordinary increase in the population of H. nodiflorum, which led to the colonization of the area where H. bermejoi had been introduced. In addition, H. nodiflorum shows remarkable ecological and phenotypic plasticity. Its most extreme plastodemes are creeping forms that occupy moist but not flooded soils. These dwarf forms were those that entered into physical contact with H. bermejoi individuals, causing the hybridization event. H. × clandestinum is very vigorous both in nature and in culture due to its size and its ability to form large lawns thanks to its ability to form thick stolons. In addition, we have also verified that it can produce viable seeds. Therefore, it was considered a severe threat to H. bermejoi, since the hybrid seemed to have greater fitness, allowing it to compete successfully for space and to contaminate H. bermejoi genetically. Hybridization is a natural phenomenon that may be a driver of plant evolution (Riesenberg 1995; Weeks et al. 2011; Piett et al. 2015), and it has even been suggested, but with great caution, that hybrids would contribute to preserve part of genetic diversity for endemic high-elevation plant species under current climate change (Gómez et al. 2015). In fact, in the Balearic Islands, several hybrids of endemic species with others with a wider distribution have been described: e.g., Lotus × minoricensis (Conesa et al. 2006) and Rhamnus × bermejoi (Fraga and Rosselló 2008). However, hybridization may have a deleterious effect when it affects rare or geographically restricted species and occurs, as in this case, by human causes (Rhymer and Simberloff 1996; Weeks et al. 2011). In fact, the risk of hybridization as a consequence of benign introductions or translocations has been described as very serious for the conservation of endangered plant species (Akeroyd and Jackson 1995; Allendorf et al. 2001; Levin 2002; Maschinski et al. 2012; IUCN/SSC 2013; Escudero and Iriondo 2003; Rhymer 2008).

Management of hybrid and introduced subpopulations Hybridization among plant species caused by conservation actions has rarely been documented (e.g., Hodder and Bullock 1997; Ferrer and Laguna 2012) despite being a known risk (IUCN/SSC 2013). For this reason, there is not much previous experience on how to manage this problem and the appropriate sequence of decisions. The first decision that had to be made was whether to conserve the new hybrid in nature. The dilemma to face was between not intervening and instead studying an extraordinary event such as the formation of a new species or intervening drastically to avoid the risk to the conservation of an endangered species. The decision had to be made in a very brief period, less than a week, since the

13

plants were at the beginning of fruit ripening. Although we had little information at that time (even without molecular confirmation that it was indeed a hybrid), we considered it a major priority to avoid any risk to the species that was wanted to be protected. It was assumed that the presence of the hybrid in nature was a very high risk and that if the H. bermejoi subpopulation was maintained at that site, hybridization could occur again in the future. On the other hand, hybridization was confined to a small area of contact between the two species, and eradication was feasible, but waiting to have more data and opinions could make eradication impossible through the dispersal of fruits of uncertain origin. Consequently, according to and in collaboration with the environmental authority, it was decided to eliminate the hybrid individuals (preserving some ex-situ ones), the entire subpopulation of H. bermejoi created in 2008 and the part of the subpopulation of H. nodiflorum that could be contaminated. Furthermore, this option is recommended for similar cases in the literature related to species introductions (Allendorf et al. 2001; Rhymer 2008 but see Ferrer and Laguna 2012).

Conclusion It is important to note that the early detection of H. × clandestinum was essential for its successful eradication, which was made possible by the regular monitoring of the introduced subpopulation. This case shows the major importance of the continuous monitoring of any introduced subpopulation. It also shows the risk that exists when making introductions outside a technically guaranteed program and without the guarantee of monitoring. At this point, it is worth highlighting recent news about the existence of a new subpopulation on the north coast of Menorca (Fraga, personal communication). This subpopulation, consisting of fewer than 20 patches in November 2016 (personal observations), corresponds to an uncontrolled introduction and shows that these sorts of actions can represent a serious threat to H. bermejoi due to its ability to hybridize. Finally, hybridization is also a risk for the ex-situ conservation of H. bermejoi. In this sense, it is important to review the existing populations in botanical gardens to take this threat into account.

Acknowledgements This work was partially funded by a grant from the Institut Menorquí d'Estudis. We are grateful to Consell Insular de Menorca, Natural Park of S'Albufera des Grau, and Direcció General de Biodiversitat i

14

Espais Natural del Govern de les Illes Balears for collaboration in the field work, and to Sequencing Service of Plant Molecular and Celular Biology Institute (Valencia, Spain) for the DNA sequencing procedure. The authors also thank Dr. Arantxa Molins, Elisenda Oliver and Pere Fraga for their very valuable help. We are very grateful to two anonymous reviewers for their valuable contributions that have allowed to improve an earlier version of the manuscript.

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Figure captions

Fig 1. Green cover area of Helosciadium bermejoi in each subpopulation. Data corresponding to the census of June 2010 is also included (data from Cursach and Rita 2013). CNe, Cap Negre station 1; CNe2, Cap Negre station 2; PF, Punta de sa Font; MV, Mongofre Vell Fig 2. Number of patches (bars) and cover area (lines) of Helosciadium bermejoi during the periods 1999-2002 (source: Mus et al. 2003), 2007-2010 (Rita and Cursach 2013) and 2015-2016 (data from this study) at the original locality. The data refer to the period of maximum vegetative development (May/June). Cover area from 2015 and 2016 corresponds to the total cover area (i.e., including dry cover area)

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Fig 3. 50% majority-rule consensus tree (of the 18 shortest trees) from ITS1 sequences using maximum parsimony analysis (371 steps). Helosciadium bermejoi sequences match with GenBank accession (64% BS) and Helosciadium. nodiflorum accessions were placed outside GenBank data but species cluster is still present (95% BS)

Fig 4. 50% majority-rule consensus tree (58 steps) from rps16-trnK intergenic spacer sequences using maximum parsimony analysis. Three hybrid individuals (H1, H3 and H4) were placed in Helosciadium nodiflorum cluster (100% BS) and one hybrid individual (H2) was placed in Helosciadium bermejoi (100% BS)

Table 1. Data of Helosciadium bermejoi, Helosciadium nodiflorum and Helosciadium. x clandestinum populations analyzed. Individuals were sampled in the island of Mallorca (Ma) and Menorca (Me). Status of plant material used for molecular analysis is indicated for each species and population Table 2. Population monitoring of Helosciadium bermejoi (census performed from June 2015 to June 2016). CNe, Cap Negre station 1; CNe2, Cap Negre station 2; PF, Punta de sa Font. In brackets, it is indicated the green cover area Table 3. Species-specific markers and mutation positions of ITS and rps16-trnK intergenic spacer regions. Lengths of sequences are 580bp and 830bp respectively. Ambiguous positions and maternal inhered bases of putative hybrids are shown in bold. Uninformative 109 position is also compared with other GenBank accessions

Figures

23

200 180 160 140 120 100 80 60 40 20 0

250 Cap Negre (station 1) 200 150 100 50

250

Area of occupancy (dm2)

No. of patches

Fig 1

200

150

100

50

0 1999

2000

2001

2002

2015

2016

0

2007

2008

2009

2010

Fig 2

24

Fig 3

25

Fig 4

26

Table and Table Captions

Taxon

Location

Sample Plant material status Code

Mongofre Vell (Me)

MV-B

Silica gel dried / Fresh

N39°59′44.4” E4°13′40.0”

Cap Negre (Me)

CNe

Silica gel dried

N39°53′48.4” E4°18′09.4”

Estellencs (Ma)

ES

Fresh

N39º39’11.0” E2º29’02.6”

Mongofre Vell (Me)

MV-N

Fresh

N39°59′40.6” E4°13′54.6

Barranc d’Algendar (Me) BG

Fresh

N39º58’31.5” E3º57’52.8”

Torrent of Sa Boual Nova TOR (Me)

Fresh

N39º56’46.1” E4º13’42.0"

Fresh

N39°59′44.4” E4°13′40.0”

Helosciadium bermejoi

Helosciadium nodiflorum

Helosciadium x Mongofre Vell (Me) clandestinum

MV-H

Site

Table 1. Data of Helosciadium bermejoi, Helosciadium nodiflorum and Helosciadium x clandestinum populations analyzed. Individuals were sampled in the island of Mallorca (Ma) and Menorca (Me). Status of plant material used for molecular analysis is indicated for each species and population

June 2015 September 2015 January 2016 June 2016 Helosciadium bermejoi No. of Total cover No. of Total cover No. of Total cover subpopulation No. of Total cover 2 2 2 2 patches area (dm ) patches

area (dm ) patches area (dm )

patches area (dm )

CNe

81

227.76 (72.46)

56

28.12

125

114.84

95

234.24 (71.87)

CNe2

1

4.7

1

3.15

5

12.36

3

12.81 (10.56)

CNo

1

4.33

2

0.038

15

2.73

3

4.63

27

PF

171

362.07

74

129.47

174

146.39

113

106.92 (77.20)

Table 2. Population monitoring of Helosciadium bermejoi (census performed from June 2015 to June 2016). CNe, Cap Negre station 1; CNe2, Cap Negre station 2; PF, Punta de sa Font. In brackets, it is indicated the green cover area

28

Taxon

Helosciadium bermejoi

Location (n=sample) Cap Negre (2) Mongofre Vell (4) RBG Edimburgh cult.

nrDNA ITS position 12 A A A

69 T T T

89 A A A

109 G G G

MF598285 MF598282 AY353979

cpDNA rps16-trnK intergenic spacer position 13 85, 86 177 418 429 C AT T C G C AT T G C no data

509

GenBank accession

C C

MF598284 MF598281

A/-

MF598293 MF598295 MF598297

C

--

A

T

G

C

MF598292 MF598294 MF598296

G

A/-

MF598299

C

AT

T

C

G

C

MF598298

G G G G T T T T T T

A A A A -

MF598289 MF598287 MF598290 MF598291 KP871514 AY360240 EF177709 AF164823 DQ005661 AY353980

T C

AT --

T A

T C

MF598288 MF598286

C C C C

AT AT AT AT

A A A A

T T G T no data no data T G T G T G T G no data no data

C C C C

KP871507 EF367705 EF185223 EF367704

Helosciadium x clandestinum

Mongofre Vell (3)

W

K

R

G

Helosciadium x clandestinum

Mongofre Vell (1)

W

K

R

Estellencs (2) Mongofre Vell (1) Barranc d’Algendar (1) Torrent (1) Cambridgeshire, UK Vaucluse, France IUIC, Illinois, France cult. Wadi Al-Yabis, Jordan Saudi Arabia Granada, Spain

T T T T T T T T T T

G G G G G G G G G G

G G G G G G G G G G

Helosciadium nodiflorum

396 -

GenBank accession

Table 3. Species-specific markers and mutation positions of ITS and rps16-trnK intergenic spacer regions. Lengths of sequences are 580bp and 830bp respectively. Ambiguous positions and maternal inhered bases of putative hybrids are shown in bold. Uninformative 109 position is also compared with other GenBank accession

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