UNIVERSITÁ DEGLI STUDI DI CAGLIARI
Dipartimento di Scienze della Vita e dell’Ambiente
Scuola di Dottorato in
Ingegneria e Scienze per l’Ambiente e il Territorio
Corso di Dottorato in
Scienze e tecnologie della Terra e dell’ Ambiente
Coordinatore: Prof. Pierfranco Lattanzi
Ph.D. Research Proposal
REPRODUCTIVE BIOLOGY STUDIES OF THE NARROW ENDEMIC
TAXA BELONGING TO THE CARYOPHYLLACEAE FAMILY
Candidate: Anna Nebot Escrigues
Tutor: Prof. Gianluigi Bacchetta
Co-tutor: Dr.ssa Donatella Cogoni
INTRODUCTION .......................................................................................................................................................... 3
CARYOPHYLLACEAE .............................................................................................................................................................. 3
Dianthus sylvestris group ........................................................................................................................................... 4
Silene mollissima group ............................................................................................................................................. 4
AIMS AND OBJECTIVES ............................................................................................................................................... 5
Dianthus morisianus Vals. .......................................................................................................................................... 6
Silene ichnusae Brullo, De Marco & De Marco f. ........................................................................................................ 7
Silene velutina Loisel. ................................................................................................................................................. 7
Floral Phenology and biology ...............................................................................................................................................9
Breeding system ...................................................................................................................................................................9
P/O .....................................................................................................................................................................................10
Reproductive success ..........................................................................................................................................................10
Inbreeding depression ........................................................................................................................................................11
Germination test ................................................................................................................................................................11
EXPECTED RESULTS ....................................................................................................................................................12
PRELIMINARY RESULTS ..............................................................................................................................................12
REFERENCES ..............................................................................................................................................................13
Introduction
There is a widespread consensus that studies on the reproductive biology of endangered, rare or
threatened species may be useful for understanding why they are endangered, rare or threatened
(Schemske et al., 1994). Depending on the reproductive biology and population history of the
species, reduced pollinator service may have several negative impacts on the plant population,
including reproductive failure (Jennersten, 1988). Reproductive biology has a direct effect on wild
populations because reproductive success and certain features of the life cycle (longevity,
dimension, and frequency of reproduction) determine population growth, while the vital form and
breeding system influence the level of genetic diversity and distribution in and among the
populations (Hamrick and Godt, 1996).
Studies on the breeding system of a plant species can provide indirect information on
conservation efforts such as patterns of genetic diversity, threats of diversity loss, risk of
inbreeding depression, and risks associated with changes in pollinator abundance or effectiveness
(Nell 2002). This is linked to reproductive success and it shows important consequences on the
viability of populations (Evans,2003 and Rymer et al., 2005Therefore, to know the breeding system
of endangered plants can help us to predict their survival capacity and projecting conservation
plans (Weller 1994, Rodriguez-Perez, 2005)
This study concerns three narrow endemic species growing on coastal systems of
southwest, northeast and northwest Sardinia. Specifically, I will be investigate the breeding system
of Dianthus morisianus Vals., Silene ichnusae Brullo, De Marco & De Marco f. and Silene velutina
Loisel., all three of them belonging to the Caryophyllaceae family.
Caryophyllaceae
The Caryophyllaceae family includes 86 genera and 2200 species, distributed mainly in temperate
regions of the Northern hemisphere The Mediterranean area, one of the world biodiversity
hotspots, in which about 13.000 endemic taxa have been recorded (Myers et al., 2000), is an
important center of diversity of this family. (Iamonico, 2013).
The Italian Flora includes 376 Caryophyllaceae taxa (34 genera), of which 92 (belonging to 13
genera) are endemics (Conti & al., 2005; Peruzzi et al.,2012). At the genus level, the
Caryophyllaceae is the sixth richest family in Italy (Peruzzi, 2010), while at species level it is the
third one (Peruzzi & al., 2012).
Caryophyllaceae are mostly annual, biennal or perennials herbs. Generally this family is
characterized by leaves which are usually opposite and decussate, more rarely alternate or
verticillate, simple, entire with or without scarious stipules. It presents the flowers in stems,
tetrameric or pentameric flowers, hermaphroditic. The flowers may be solitary, or are arranged in
branched inflorescences of various kinds. It presents 8-10 stamens and 2-5 styles, superior ovary
with 1,3-5 incomplete septa. The fruit is usually a dry capsule that releases its seeds by opening at
the tip. Capsule fruit, dehiscent by apical teeth or shells and equal or double at the number of
styles. Seeds are reniformed or subglobosed. (Castroviejo et al., 1990)
Dianthus L. and Silene L. genus comprise the higher number of endemics plants in Italy, with
27 and 26 taxa respectively (about the 29.35% and 28.26% of the total endemics
Caryophyllaceae). (Iamonico, 2013). The 28.14% of the endemics occurs in Sicily and Sardinia.
Dianthus sylvestris group
Within the genus Dianthus, the D. sylvestris Wulfen group can be considered as one of the most
complex, and it is still not deeply investigated.
This group is represented by Dianthus arrosti C. Presl, D. siculus C. Presl, D. graminifolius C.
Presl, D. cyathophorus Moris, D. gasparrinii Guss., D. longicaulis Ten., D. virgatus Pasquale, D.
tarentinus Lacaita, D. morisianus Vals., D. japygicus Bianco & Brullo, D. sardous Bacch., Brullo,
Casti & Giusso, D. busambrae Soldano & F. Conti, D. brachycalyx Huet sp. nov., D. oliastrae sp.
nov., D. insularis sp. nov., D. genargenteus sp. nov. And D. ichnusae sp. nov. Besides, two new
subspecies are recognized within D. ichnusae (subsp. ichnusae and subsp. toddei). (Bacchetta et al,
2010)
Silene mollissima group
The species belonging to Silene mollissima (L.) Pers. group represent a very ancestral taxa
occurring in the West Mediterranean area (Keifer & Boquet 1979; Jeanmonod & Boquet 1981;
Brullo & Signorello 1984; Jeanmonod 1984). They are chasmophytes with scattered and often
punctiform distribution, growing in the rocky places near the sea or in the coastal and mountain
cliffs.
This group includes seven endemic species distributed in the western mediterraneal sea
and live on cliffs of limestone mountains or in the coast. This group is composed by S. mollissima
(L.) Perso (endemic of Mallorca and Menorca), S. andryalifolia Pomel (grows in north of Africa and
South Spain), S. auriculifolia Pomel (endemic of Orano (Algeria)), S. tomentosa Otth (endemic of
Gibraltar), S. hifacensis Rouy ex Willk (endemic of Ibiza and Alicante), S. hicesiae Brullo et
Signorello (endemic of Panarea) and S. velutina (Jeanmonod, 1984). S. ichnusa was found in 1996
and it was introduced in this group.
Aims and objectives
The objectives of the research project to conduct during the PhD program are:

To know the current status of these species regarding the reproductive success.

To assess the effects of the phenological components on its reproductive success

To analyze if these species are threatened by anthropic factors or also by natural
factors (decrease pollinators, inbreeding depression, auto incompatibility, etc.)

To evaluate if there are differences in reproductive phenological patterns between the
natural plants and cultivated introduced plants
Study species
Dianthus morisianus Vals.
D. morisianus is a perennial suffrutex characterised by numerous woody stocks and erect
stems 20-45 cm long, and by a basal rosette with thin and linear leaves, 1-15 cm long. The stems
bear terminal multi-flowered heads (normally 2-18 flowers/head), the calyx is characterized by
lanceolate teeth and the color of the corolla is normally pink. The flowering season is from early
May to late June, and ripe fruits can be found during June–July (Bacchetta et al., 2010). This plant
grows on stabilized dunes at the edge of Juniperus spp. micro-forests and scrub dominated by
Cistus spp.
D. morisianus is the only psammophilous species of the genus Dianthus in the
Mediterranean basin. It is found in only one population, on the Portixeddu coastal dune system in
Buggerru, south-west Sardinia and it is one of the most threatened plants on the island (Bacchetta
et al., 2012).
The natural habitat of D. morisianus has been strongly modified by human activities,
causing habitat loss and fragmentation: there are several settlements in the species’ habitat and
since 1950 much of the dune system has been afforested to stabilize the dunes and halt the
movement of sand inland (Cogoni et al., 2013). The small size of the population and the limited
seedling recruitment make D. morisianus potentially prone to extinction, and it is categorized as
Critically Endangered on the European (Bilz et al., 2011) and Global Red Lists (Fenu et al., 2011).
Because it is one of the most threatened plant on the island (Bacchetta et al., 2012), the
Autonomous Region of Sardinia funded a conservation project for D. morisianus. The project
comprises in situ and ex situ research and experimental projects, such as the construction of
protective fences (Fenu et al., 2012) and the reintroduction project.
Detailed investigation of the population ecology of the existing population of this species,
with the aim of identifying the constraints on its viability, have been done with another PhD thesis.
Specifically, were analysed some critical stage of its life-cycle (i.e. modeling of emergence and
germination), was evaluated the phenological pattern and the conservation status. (Cogoni, 2012)
In this PhD project D. morisianus will be studied in two populations in the field (the natural
one and the reintroduction one) and in cultivated plants in the Botanic Garden at Cagliari.
Specifically, for the reintroduction 113 plants, grown from seed collected ex situ from the wild
population, were reintroduced in a specific site. The site was chosen through preliminary research
focused on the ecology of D. morisianus. These surveys facilitated the identification of a suitable
area c. 150 m from the natural population, in a protected site, managed by public administration
(EFS, Ente Foreste della Sardegna; Cogoni et al., 2013). Plant cultivated ex situ, at the in the
Botanic Garden of Cagliari, came from fruits collected from the wild population
Silene ichnusae Brullo, De Marco & De Marco f.
Is a perennial small shrub, tomentose with hairs, woody stock robust, with caespitose sucker,
few branched provided with dense and compact persistent rosettes. Flowering stems robust, erect,
solitary 2-3 mm wide, with 3-6 not flowering nodes and internodes rigid 4-8 cm long. Basal leaves
persistent moderately thick, narrowly elliptical or lanceolate-elliptical, 4-14 x 1-3 cm, hairy with
hairs 0.5-1 mm long, shortly connate, acute or subacute, with petiole (2) 2.5-4.5 mm wide, midrib
prominent with lateral (Brullo et al 1997).It is a chasmophyte localized at 40-90 m altitude in the
north-westem slope of Capo Falcone (N-W Sardinia), on siliceous rocks near the sea. It is a
member of a dwarf-shrub community characterized by spiny cushion-like endemics, such as
Astragalus terraccianoi, Centaurea horrida and Genista Corsica. Some plants grow on the ledges of
the cliff as well. The flowering season is from May to early June, and the fructification is in June.
Currently, other studies focused on the micro and macro ecology of this species are being
conducted.
This species will be studied in the only known natural population in the field and one ex-situ
population (in the Botanic Garden of Cagliari). In the field will be studied 20 plants and in the
Botanic Garden 30 plants.
Silene velutina Loisel.
S. velutina presents woody stock covered residues from foliar rosettes with sterile and
fertile stems arcuate-erect (25-60 cm) with numerous internodes (5-12) below the inflorescence,
loosely caespitose, pubescent leaves with moderately crassulente. Leaves of rosettes, attenuated
into a petiole slightly guainante, strictly elliptic and acute densely velvety pubescent on both
sides.
It is a coastal Corso-sardo endemism distributed in nearly 30 sites with a relatively low number of
individuals. In Sardinia it is located in some granitic islets of La Maddalena archipelago. It lives in a
small community where predominates over Lavatera arborea L., Allium ampeloprasum L., Allium
commutatum Guss. e Anthemis maritima L. The flowering season is from late May until July, and
fructification starts in June.
This species is afected by invasive species (Carpobrotus and Opuntia), predators (rats), and
decrease of habitat due to tourism activities. The UICN classified this species as Near threatened
(Buord et al., 2011).
It is catalogued as a priority species and included in the Appendix II of the Habitats
Directive and Appendix I of Bern Convention (Convention on the Conservation of European
Wildlife and Natural Habitats). In Sardinia it is included in the Parco Nazionale La Maddalena.
Currently, other studies focused only on the micro and macro ecology of this species are being
conducted.
This species will be studied in the Abbatoggia population in the field and in the ex-situ
population (at the Botanic Garden of Cagliari). In the field will be studied 20 plants and in the
Botanic Garden 30 plants.
Material and methods
Floral Phenology and biology
To analyze floral phenology and biology, daily observations will be carried out. All the open
flowers, at the moment of anthesis, will be tagged. In order to study the different stages from
anthesis to fructification, a flower per plant will be tagged. These flowers will be used for control
treatment for which no hand pollination experiment must be done; only to know the moment of
anthesis and to harvest the seeds is necessary. These flowers allow us to compare the results of
natural pollination with the hand pollination experiments. This study will be repeated in May and
June/July to know if there are any differences in the duration of flowering.
With these dates can be calculated:
 The flower intensity (number of flowers opened each day)
 The flowering duration (individual and total)
 The maximum flowering moment (the number of days from the first open flower in the
population until the day of maximum peak on each plant)
 Flowering synchrony (the number of days when the flowering of one individual overlaps
with the flowering of every other plant in the population)
Every day I will observe the tagged flowers in order to know in which stage they are. Then, I will
cut 5 flowers in the same stage of flowering control to analyze it in the lab. To characterize each
stage, I will measure the flower diameter, peduncle, calyx, petal limb, stamens and styles of these
flowers.
There are several works that show that when the flowers are pollinated they are withered
first to save resources. To know if these species presents this characteristic, I will study the
duration of flowering in the flowers used in the pollinated experiments, to compare the result with
non-pollinated flowers.
Breeding system
The mating system will be studied through 6 pollination experiments. The treatments will be:

Spontaneous auto pollination: this experiment consists in putting a fine mesh bag
around a flower before the anthesis. These bags are not taken away until the moment
of collecting the seeds. This experiment allows us to know if the flower can auto
pollinate without pollinators.

Self hand-pollination:it consists of pollinating the flower with pollen from another
flower of the same plant. These flowers will be bagged with a fine mesh bag and every
day the stamens will be cut to avoid the auto-pollination.

Cross hand-pollination:this experiment consists of pollinating flowers with pollen from
another plant of the population.

Control: these flowers will be tagged but not bagged to analyse the natural pollination.

Flowers are bagged with a mesh that allows the pass of wind but not the pollinators.

Supplementary pollination. To know if the natural pollination is sufficient or not to
produce all the seeds.
P/O
The Cruden’s index (P/O) is a predictive system to know which is the reproductive system in the
study species from the number of primordia and the numbrer of pollen grains. (Cruden, 1977).
One anther per plant and an ovary per plant must be harvest. In ex situ population, these
experiments will be repeat in May and in July. The anthers and ovary will be fixed with ethanol. To
analyze the number of pollen grains and ovule quantity, will be use the methodology proposed by
Dafni et al., 2005 with some modifications (Cursach & Rita, 2012). Each anther will be preserved in
an eppendorf with 1ml of the fixing liquid. To open the anthers and release the pollen grains a
vortex will be used. Then, 10 sub-samples of 10 μl per each anther will be scored. The grains of
pollen will be counted with the help of microscope. After this, the result will be extrapolated with
the total volume (1ml). Then, this value will be multiplied by the number of anthers per flower.
To count the number to primordia per flower, the ovaries will be dissected with a magnifying
glass, and then, the number of ovules per each ovary will be counted.
Reproductive success
When the fruits are mature they are harvested and kept in envelops (one per fruit). Then,
the effectiveness of each treatment is compared in terms of fruit set (proportion of treated
flowers that eventually produced a fruit)- Alonso & Garcia-Sevilla,2013-, seed set (number of seeds
regarding number of ovules per flower)-Carrió et al., 2008-and seed weight.
With these values the ISI index can be calculated (index self incompatibility). Values greater
than 1 indicate that the species is autocompatible, values from 0.2 to 1 indicate that this species is
partially autocompatible and values minor that 0.2 indicates that this species is autoincompatibile;
and, IA index (autonomus self pollination), values greater than 1 indicate that the species presents
autogamy, values from 0.2 to 1 indicate that this species is partially autogamy and values minor
that 0.2 indicate that this species presents autocompatibility but have mechanisms which prevent
the autopollination. (Ruiz-Zapata & Arroyo, 1978).
Inbreeding depression
Inbreeding depression value is calulated with the results of breeding system and
germination tests, using fruit set, seed set, seed weight, T50 (days from planting until a 50% seeds
were germinated) , percentage of germination, and seedling growth, as δ= 1-(Ws/Wo) –
Charlesworth & Charlesworth 1987. Ws is the mean production with self-pollination, and Wo, with
cross-pollination. We obtained accumulative value of inbreeding depression with the relationship
of the six samples of dates. The final values are zero to one. Zero: don’t have inbreeding
depression, positive values represents more production of seeds with cross-pollination than the
self-pollination, the negative values the opposite. The average level of inbreeding depression was
also calculated as δ= 1-[(Wsf/Wof) x (Wss/Woss) x (Wsw/Wow) x (Ws%/Wos%) × (Wst50/Wot50)
× (Wsg/Wog)].
Germination test
To know the degree of germination seeds from each treatment, three replicates of 25
mature seeds on agar Petri dishes will be placed. Seeds will be incubated in a chamber at optimal
conditios for each species and monitoring daily. Every day the dish with germinated seeds will be
checked and removed until last germination or 30th day. On this way, the percentage of
germination, and T50 (the necessary time to have 50% of seeds germinated) -Coolbear et al.,
1980-, will be calculated.
Expected results
In the first year the in situ and ex situ populations of D. morisianus and in situ population of S.
ichnusa and S. velutina will be studied and. Through these studies I will determine the breeding
system of these species which will show if they are threatened by genetic factors or external
factors.
Preliminary results
The first thing that I did was transplant the D. morisianus from pots of 7Litres to pots of 11L.
This allowed D.morisianus to grow in size and to produces more stems.
Then, I put to germinate seeds from D. morisianus, S. ichnusae and S. velutina, in order to
measure the growth rate. These plants of S. ichnusae and S. velutina will be used for the studies of
ex-situ populations.
Until now, it seems that the flowers pollinated with the pollen of the same plant (self) and
for the other plant (cross) can pollinated and the fruit begins to form.
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