First insular population of the critically endangered Cistus heterophyllus subsp. carthaginensis on Cabrera Archipelago National Park (Balearic Islands, Spain)

Cistus heterophyllus subsp. carthaginensis is considered one of the most endangered plants of Spain and has been object to many conservation programs and research projects. Until now, only two small spots were located in Europe: a population of one self-incompatible specimen was found in Valencia and another small population in Cartagena, disturbed by human impact and hybridization with close-relatives. Therefore, the conservation of this taxon has been one of the most defeating issues in the current plant conservation strategies of the Mediterranean workgroups. In this study, we report, describe and cense a new population of this taxon located at the furthest accessible area of the isle of Cabrera Gran in Cabrera Archipelago National Park. The population contains 59 individuals of C. heterophyllus subsp. carthaginensis, the most abundant known population of this species, with a high representation of young individuals (83%). However, some threats are currently compromising the stability of this population: the presence of Cistus albidus in surrounding areas is producing hybrids (Cistus × clausonii) and the high abundance of rats and rabbits is affecting the population recruitment. For this reason, urgent conservation actions must be designed to protect and enhance this population. Overall, this new finding offers new insights on the distribution and conservation strategies of this species.


The discovery and conservation of Cistus heterophyllus in Europe
Cistus heterophyllus Desf. is a species endemic to the Western Mediterranean basin, with two highly threatened populations in the Iberian Peninsula. These European populations have been described under the subspecies carthaginensis (Crespo and Mateo 1988;Demoly and Montserrat 1993). This species lives in thermomediterranean scrublands with low annual precipitation regimes (Crespo and Mateo 1988).
At the beginning of the twentieth century, an abundant presence of an unknown rockrose was cited at the area of Cartagena (Peña del Águila and Sancti Spiritu) for the first time, which was studied by Carlos Pau and identified as Cistus polymorphus Willk. (Jiménez 1903). Nevertheless, after the publication of a monograph on the genus Cistus (Grosser 1903), Pau decided to describe the material as a new taxon: Cistus carthaginensis Pau (1904). Subsequently, this species was ascribed to the North African taxon described as C. heterophyllus Desf. (Vicioso 1946;Martín Bolaños and Guinea 1949). In the early 1970s, the taxon was considered extinct from the Iberian Peninsula (Esteve 1973) as no plants were detected anymore, until in 1986 a single individual was found in La Pobla de Vallbona (Valencia) and the subspecies C. heterophyllus subsp. carthaginensis (Pau) Crespo and Mateo (1988) was formerly proposed. This Valencian specimen did not produce fruits due to self-incompatibility, common in the genus Cistus (Boscaiu and Güemes 2001).
In 1993, a small population of 9 individuals was discovered in Murcia near Cartagena (Robledo et al. 1995), but in 1998 the area was devastated by a fire and the species disappeared. The following year, the species germinated from the soil seedbank and 26 individuals successfully colonized the area (Sánchez-Gómez et al. 2018). These individuals presented a certain hybridization with Cistus albidus, which is very abundant in the area and raised a significant risk factor for the conservation of this taxon (Navarro-Cano 2018a), being the Valencian plant much genetically purer than the Cartagena plants (Jiménez et al. 2007). These hybrids between C. albidus and C. heterophyllus are abundant in North Africa and have been described as Cistus × clausonii (Jiménez et al. 2007;Navarro-Cano et al. 2009). The vigour of these hybrids is indicative of their possible evolutionary relevance (Fernández-Mazuecos and Vargas 2018).
In 2012 the Valencian plant was able to produce an unique fruit due to an uncommon self-pollination event, but only one seed was viable to reproduce (Chenoll et al. 2020). In 2013, the individual fructified again and 142 seeds were obtained, from which several specimens were grown (Ferrer-Gallego et al. 2018). In 2022, the death of the original Valencian specimen was confirmed, but more than 1500 plants and 10,000 seeds were finally obtained (Ruiz 2022). Because the origin of the current population comes from a single individual, a low genetic diversity is expected, compromising the stability of the population in the long-term.
Cistus heterophyllus subsp. carthaginensis is listed in the Spanish Catalogue of Threatened Species (Royal Decree 139/2011) in the highest category of threat "En Peligro de Extinción". In 2018 it was considered in critical status (Order TEC/1078/2018 being the unique plant species to be declared in high risk to disappear. In the Region of Murcia, it is included in the Regional Catalogue of Protected Wild Flora of the Region of Murcia (Decree 50/2003). At the same time, it is included in the Valencian Catalogue of Threatened Flora Species (Decree 70/2009). In both cases, C. heterophyllus subsp. carthaginensis is categorized as "En Peligro de Extinción". There 1 3 are also Recovery Plans approved for the Iberian populations, both in Murcia (Decree 244/2014) and in Valencia (Order 1/2015). Also, as a result of being legally declared in critical situation and in order to optimize and coordinate conservation strategies implemented to this species, a Working Group formed by state administrators and technicians from Murcia and Valencia was created in 2018 and currently ongoing. Since the discovery presented in this study, Balearic technicians have also joined to the Working Group.

The vegetation of Cabrera Archipelago National Park
The Maritime-Terrestrial National Park of Cabrera Archipelago (Parque Nacional Marítimo-terrestre del Archipiélago de Cabrera), located at the south of Mallorca, was officially declared in 1991 and consists of several islands and islets. The largest island is Cabrera Gran, with an area of 11.54 km 2 . It has an annual rainfall range from 330 to 407 mm, an average temperature of 18 °C and a semi-arid thermo-Mediterranean climate. Its highest peak is in Puig de na Picamosques (172 m). The Park's Natural Resources Management Plan (Royal Decree 1431/1992) limits human intervention on the vegetation in order to allow their spontaneous evolution and to provide model communities for the study of the ecological processes linked to them.
The flora of the Cabrera Archipelago was studied by Marcos (1936) for the first time, followed by a review of Palau (1976) including more details of many taxa. In the early 1990s, an exhaustive plant species inventory of all 500 × 500 m UTM grids of the whole archipelago was carried out, published as part of a monographic work of Cabrera Archipelago Rita and Bibiloni 1993). Also based on this data, a guide with an analysis of the biodiversity of the archipelago was published (Bibiloni and Rita 1995). Recently, an update of this monograph has been published with several chapters dedicated to flora and vegetation Traveset and Rita, 2020;Seguí et al. 2020) and currently, promoted by the staff of the National Park, there is a proposal to carry out a new update of the flora from the entire archipelago.
Until now, in Balearic Islands, the genus Cistus L. was represented by 5 taxa (Demoly and Montserrat 1993): C. albidus L., which is present on all islands, Cistus creticus L., that lives only on Menorca, Cistus monspeliensis L., existing on all islands; Cistus salviifolius L., present on all islands except Dragonera and Cabrera, and Cistus clusii subsp. multiflorus Demoly, present on Mallorca, Ibiza, Formentera and recently extinct from Cabrera (at the coast near the shore of l'Olla).
We present our finding of a new population of the threatened C. heterophyllus subsp. carthaginensis in the northern part of the island of Cabrera Gran. Therefore, this is the first population stablished in insular conditions and also under the protection of National Park laws and management. This finding represents an important extension of the distribution of this endangered species (Fig. 1) and new insights to disentangle its phylogeography.

Material and methods
The discovery process of this population is explained below. After this finding, we proceed to explore the surrounding area (radium of ca. 500 m) attempting to expand the area of occupancy of the population. All individuals were geolocated by GPS (distance error of 3 m) and manually marked using the official labels used by the environmental authority to register threatened plant species in Balearic Islands. From each individual, we obtained information about their approximate life stage depending on their size and reproductive capacity (seedlings: individuals of less than one year old; juveniles: those of more than one year old but not reproductive yet; and reproductive matures: adult plants that already flowered and fructified). Also, we registered dead individuals and those showing predation signals.
In order to confirm the taxonomic identity of this species, we obtained plant material from the largest individual (in order to minimize sampling impact on the threatened species) and we observed and described all morphological traits necessary to recognise the taxon. Detailed pictures of the most important plant features (e.g., trichomes and seeds) were made using an optical microscope coupled to Canon 90d camera, using WeMacro's automatic rail and the software Helicon focus v8.1.0 stacking software (Helicon Software 2022) to merge stacks into a fully focused images.
After fieldwork, we introduced all geopositioned data on KML format in QGIS v3.10. (QGIS Development Team 2020) and they were mapped using Balearic Islands shapefile obtained from the Cartography Service of the Government of the Balearic Islands.

The discovery of Cistus heterophyllus subsp. carthaginensis in Cabrera Archipelago
In the early 1990s, Gabriel Servera, a National Park guard, detected the presence of a species of Cistus on the island of Cabrera Gran that could not be ascribed to any of the species known in Mallorca, giving the identity of Cistus cf. creticus. Two decades later, in September 2015, the Forestry Centre of the Balearic Islands collected seeds of this unknown taxon. After observing the specimens, we confirmed that it was not Cistus creticus although the definitive identification remained unsolved. We were able to detect some individuals having similar traits to C. albidus, which led us to think that the unknown taxa could originated by hybridization. We revisited the island of Cabrera Gran in April 2022, coinciding with the flowering season of the species, favouring its identification as C. heterophyllus, mainly due to the yellow macules with white upper part present at the base of the petals, which quickly distinguish it from other pink-flowered Cistus species present in the Western Mediterranean Basin (Fig. 2A).
A more detailed analysis allowed us to determine with certainty that the individuals found in Cabrera Gran also presented the morphological traits associated to the subspecies C. heterophyllus subsp. carthaginensis. In addition, the intermediate individuals coincided with the description of Cistus x clausonii (Cistus albidus × C. heterophyllus). Cistus albidus has been cited in different locations of Cabrera Gran (Marcos 1936;Palau 1976), but currently there are only 18 individuals in the entire Cabrera Archipelago, 6 individuals located a few meters away from the population C. heterophyllus subsp. carthaginensis, and another 12 specimens about 760 m away.
The flowers of the individuals found in Cabrera Gran have external sepals slightly mucronate and internal ones apiculate, of 41.6 mm ± 0.5 mm in diameter, seeds are spheroidal-tetrahedral (Fig. 2B), with a densely papillose external cover or with alveolate faces and papillose edges, sienna-coloured (Navarro-Cano 2018b). Indumentum of the young branchlets constituted by stellate trichomes (Fig. 2C) and very few simple trichomes (Crespo and Mateo 1988). Also, all stellate trichomes were found adpressed to leaves (Fig. 2D). Cistus albidus contains many spreading stellate trichomes while C. x clausonii present stellate trichomes of intermediate disposition (Navarro-Cano 2018b). Therefore, pure specimens of C. heterophyllus subsp. carthaginensis can be distinguished in the population. These plant features, combined with the semi-arid thermomediterranean bioclimate where this species exists in other populations of Spain and north-Africa, allow us to confirm that this threatened species was present in Cabrera Gran.
Cistus heterophyllus subsp. carthaginensis (Pau)  Cistus heterophyllus subsp. carthaginensis plants are found in the northern part of the island of Cabrera Gran, at the furthest point from the port and away from human frequented areas. The central population runs parallel to a forest track, which is not reflected in the 1980 toponymic map of Cabrera by Cosme Aguiló, indicating that it is a modern construction. After a population census, we detected a total of 59 individuals of C. heterophyllus subsp. carthaginensis. Ten individuals were more than one year old and were reproductive; 41 seedlings were less than one year old, and 8 individuals had hybrid features (C. × clausonii). However, the individuals of this species are very difficult to detect if they are not in bloom, since the plants live among other species and get confused in them (e.g., Cistus monspeliensis or Pistacia lentiscus). Probably, this difficult discrimination of the vegetative parts of the species within the plant mosaic where it is located is the reason why no botanist has detected the presence of this taxon in this area of Cabrera Gran until now.

Current status of the insular population
The population is distributed in a central core, parallel to the forest track, with 9 pure and reproductive individuals, 33 pure seedlings, and 1 hybrid; a small core about 40 m east of the central core, with 1 pure individual, 1 dead individual, and 7 seedlings; and a last core north of the central core, with 7 reproductive hybrid individuals and 1 pure seedling (Fig. 3). This last core composed of 7 hybrids is only 7 m from a small population of Cistus albidus consisting of 6 individuals. This proximity explains why all the individuals of this core are morphologically identified as hybrids.
When Cabrera Archipelago National Park was declared, the environmental authority decided to eliminate goats and sheep present on the island, whereas agricultural activities were also abandoned. This land use changes led to a notable increase in woody vegetation of Cabrera Gran , which could be the reason why C. heterophyllus subsp. carthaginensis is probably losing its habitat restricted to scrub clearings. In fact, we found most of the young plants along the open space left by the forest track, revealing a clear preference of this species for sparsely vegetated areas.
This case is not the first endangered rockrose endemic to Western Mediterranean basin that requires conservation actions. Helianthemum caput-felis (Cistaceae), which has a similar distributional range -but living at different habitats-than C. heterophyllus, is considered Endangered and extinction models predicted a quasi-extinction of 45% in three generations (Sulis et al. 2020). Also, this species have a similar lifespan and plant architecture than C. heterophyllus whose population stability rely on short-term population dynamics (Sulis et al. 2018). This case can be used as a model for further conservation strategies on C. heterophyllus.
Additionally, future studies should check the degree of hybridization of this new island population and whether this could be detrimental to the conservation of the species. The scarce presence of C. albidus on Cabrera Gran offers a very interesting scenario for further research on the phenomenon of hybridization of this species with C. heterophyllus subsp. carthaginensis. In addition, it remains to be resolved what are the phylogenetic similarities with other populations of this species and the phylogeographic pattern that frames this distribution. Most of the seedlings showed predation marks, indicating that they suffer from a high herbivory impact. For this reason, it is clear that the presence of rabbits and rats in Cabrera Gran is an important factor compromising the population recruitment and demographic stability of this species. For this reason, it is necessary to rapidly create and develop a recovery plan for this species in the Balearic Islands. Conservation actions must be taken in the near future to protect this new population of the endangered C. heterophyllus subsp. carthaginensis, which is also the very first insular population found from this species, and must be included as a new taxon from the Balearic Islands flora, a new endangered species to be included in the Red List of Balearic threatened species and to the list of the most threatened Mediterranean islands plants.