Petagnaea Gussonei: an Ancient Plant to Save

Project location: ITALY
Project start date: January 2008 - Project end date: January 2011
Project number: 2007-15
Beneficiary: Università degli Studi di Napoli Federico II

Final Report (2010-2011)
Petagnaea gussonei: an ancient plant to save

Petagnaea gussonei (Spreng.) Rauschert is a rare plant typical of the North-eastern Sicily (Nebrodi Mountains). Actually, it is considered an endangered species (Gianguzzi et al. 2004), whose biology is not effectively well known. The present study was aimed to gain more detailed informations about P. gussonei biology and genetics through the development of different phases (subprojects), whose previous results are available on the Nando Peretti web page:
On the whole, the project includes the following subprojects:
(1) "Ecological and environmental characterization of Petagnaea gussonei localities" (started in 2008 and finished in 2009).
(2) "Biochemical characterization of the Petagnaea gussonei plant materials" (started in 2008 and finished in 2009).
(3) "Ecophysiological characterization of Petagnaea gussonei" (carried out from 2009 till 2010), whose findings are here reported. .
(4) "Molecular characterization of Petagnaea gussonei", whose results are here discussed. This subproject concerned phylogeny of P. gussonei (a study performed from 2008 till 2009) and its genetics of population.

(3) Ecophysiological characterization of Petagnaea gussonei (second part)

A research article entitled "Photochemical activity and leaf functional traits of Petagnaea gussonei (Spreng.) Rauschert, an endangered relict Sicilian species (Apiaceae)" was submitted for publication on an international journal.

The regulation of light energy absorption is a key process in evaluating the acclimation of a plant in its habitat. So, two different groups of P. gussonei plants were studied during the second year of this research with the aim to gain informations about the response of the plants to a different exposition to light. The first group included plants grown in pots, and then always exposed to light, while the second one was made by plants growing in a open field, where light intensity was variable. No significant temperature variation existed between the groups of plants. Each group was analyzed for photosynthetic pigments content (total chlorophyll and carotenoids), specific leaf area (SLA) and leaf water content (LWC) in spring and in winter time, showing an important variation in photosynthetic pigments composition. In spring, both plants grown in field and in pots showed the highest value (P<0.01) of total chlorophyll compared to winter. On the contrary, in winter the highest amount of total carotenoids (P<0.01) was found in both groups (Tab. 1). It was evident how chlorophylls and carotenoids content was higher in plant samples collected in field as compared to the ones grown in pots. As regards to leaf traits, a higher (P<0.01) specific leaf area (SLA) was found in spring compared to winter in plants taken from both sites but plants grown in pots showed always higher values compared to the other group of plants. No difference, instead, was found in relative water content (LWC) (Tab. 1). All the findings collected showed that P. gussonei is able to respond to different light conditions with a modulation of pigments content and of a modification of SLA. This way, P. gussonei is able to modulate the capture of light energy during spring and winter. The significant increase of SLA in plant grown in pots suggests a higher photosynthetic capacity at non limiting light conditions.



Table 1 - Total chlorophyll content (Chl a+b), total carotenoid content (Car tot), specific leaf area (SLA) and leaf water content (LWA) of P. gussonei measured in spring and winter in two different sites. Values are expressed as means ± SD (n = 8).

(4) Molecular characterization of Petagnaea gussonei (second part): Population genetics

Preliminary results were submitted as part of an oral communication to the Italian Botanic Society, Floristic and Biosystematic groups Congress (Rome, 22-23 October 2010). An abstract was published in a book of the congress proceedings:

Sepe F., Paino L., De Luca P., De Castro O. 2010. Analisi della variabilità genetica in Petagnaea gussonei (Apiaceae) attraverso lo studio dei microsatelliti plastidiali e dei Polimorfismi di Lunghezza dei Frammenti Amplificati (cpSSR ed AFLP). In: La biodiversità vegetale in Italia: aggiornamenti sui gruppi critici della flora vascolare, Peccenini S., Domina G., Salmeri C. (Editori). Società Botanica Italiana. Pp. 23-24. ISBN 978-88-85915-03-9.

Finally, a manuscript entitled "Comparative genetic study in the ancient and endangered relictual Apiaceae, Petagnaea gussonii (Spreng.) Rausch." is going to be submitted to an international journal for possible pubblication.

Genetic variability of P. gussonei populations was evaluated using chloroplast microsatellites (cpSSR) and Amplified Fragment Length Polymorphism (AFLP), which are genetic markers characterized by a great variability. This analysis was aimed to better understand the evolutionary pattern of P. gussonei, giving this way a contribution to the adoption of suitable measures for its protection and conservation.
After many expeditions, 17 P. gussonei populations were identified by Prof. Lorenzo Gianguzzi (University of Palermo, Dep. Botanical Sciences) and 134 individuals were collected. Genomic DNA was extracted from all the individuals following a modified CTAB procedure (Doyle & Doyle 1990; Focus, 12: 13-15.). Ten universal primers cited by Weising & Gardner (1999; Genome, 42: 9-19) were used for cpSSRs analyses while 64 selective primers were tested for AFLPs study according to a modified protocol of Vos et al. (1995; Nucleic Acid Reserch, 23: 4407-4414).
The amplification reactions obtained by the use of 7 different combinations of cpSSR primers originated 672 fragments corresponding to 2 haplotypes, one of them found with a higher frequency (81%). An evaluation of plastidial genetic differentiation among all populations was null [Gst~0 (<0.0001)], moreover no phylogeographic structure was detected among the populations (Gst=Nst). Hs and ht values showed a moderate "intra-population" and "inter-population" genetic diversity [0.35 (±0.062) e 0.33 (±0.048)]. Finally, even the analysis of molecular variance (AMOVA) confirmed statistically what evidenced by previous analyses.
As to AFLP electrophoretic profiles, for each oligonucleotidic combination 103 loci were chosen as conformed to Lynch & Milligan method (1994; Molecular Ecology, 3: 91-99), besides to be unambiguous in their qualitative discrimination. It was evident, from the collected findings, that molecular variance (AMOVA), i.e. the genetic variability partition is actually widely expressed within the population with a value equal to 82%, confirming the results obtained by analyzing plastidial microsatellites. So, a moderate genetic variability structuration was present among populations as variability among population was just equal to 18%. Genetic variability within populations was moderately represented, as proved by the different analyzed indices whose averages were equal to 0.136 as regards to Nei index (H), and to 0.193 as far as to informative index of Shannon (I). In short, considering Petagnaea in the total, i.e. considering all the analyzed populations, it is possible to notice how this entity is going towards a genetic structuration of its populations as evidenced by the total fixation index (Φ-PT=0.18; θ-I=0.25) and confirmed by the dendrogram of the Φ-PT relations. Thus, the collected data indicate that the species is actually in a phase of transition characterized by a moderate genetic variability
Summarizing the results what will be exposed in the new article (see above), it seems clear P. gussonei is an endangered species as the exiguous number of its population, which actually have a moderate variability in both plastidial and nuclear genome.
This moderate level of differentiation supports the hypothesis that both fragmentation and isolation in Petagnaea are actually in process. Anthropic activity (agriculture and springs capitation) as like as drastic weather changes are leading more and more Petagnaea within its ecological niches.
On the whole, these data could result comfortable if compared to the analyses carried out on some other paleo-endemisms, which have not any genetic variability (Zelkova sicula, Fineschi et al. 2004; Forest Ecology and Management, 197: 273-278; Wollemia nobilis, Peakall et al. 2003; Molecular Ecology, 12: 2331-2343), thus raising up a reflection about P. gussonei species status.
Even if P. gussonei has a modest global genetic variability, it has to be protected as endangered species (classificated as ENDANGERED by I.U.C.N., International Union Conservation Nature,, surviving within a restricted area with all "possible" consequences that could derive from this situation in future (genetic derive, inbreeding depression, and so on). In the course of time, environmental changes had occurred in the area where P. gussonei lives owing to anthropic alterations. These have caused worrisome demographic variations within the species, leading till a possible extinction of it. What is actually really worrying it's the quantity, the rate and the velocity of environmental changes causing serious problems to P. gussonei survival.
Finally, it is good to remember in small populations as P. gussonei ones, additional casual factors (demographic, environmental, genetic and catastrophic events) increase the risk of extinction. In fact, a rare species, like Petagnaea, takes, by definition, a rare genome and has an addicted value above all when it is represented by a small population because it is more exposed to extinction than a species characterized by a large population.

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