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.............................................................................................................................................................3Dianthus sylvestris group..........................................................................................................................................4Silene mollissima group............................................................................................................................................4

AIMS AND OBJECTIVES............................................................................................................................................. 5

Dianthus morisianus Vals..........................................................................................................................................6Silene ichnusae Brullo, De Marco & De Marco f........................................................................................................7Silene velutina Loisel.................................................................................................................................................7

Floral Phenology and biology..............................................................................................................................................9Breeding system..................................................................................................................................................................9P/O....................................................................................................................................................................................10Reproductive success........................................................................................................................................................10Inbreeding depression.......................................................................................................................................................11Germination 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 biologyTo 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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