23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, , 26- 30 October 2008

“Dichotomous key for the identification in nurseries of the main poplar clones cultivated in European Union”

Giuseppe Nervo, Franco Picco, Achille Giorcelli

CRA – PLF Unità di Ricerca per le Produzioni Legnose Fuori Foresta

Strada per Frassineto 35 Casale Monferrato (AL) - 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

A dichotomous key is a tool that allows the user to determine the identity of items in the natural world, such as , wildflowers, mammals, reptiles, rocks, and fish.

Keys consist of a series of choices that lead the user to the correct name of a given item.

"Dichotomous" means "divided into two parts".

Therefore, dichotomous keys always give two choices in each step. Dichotomous key

step-by-step analysis of morphologic characters possibly showing two alternative ways 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

Correct identification of poplar varieties is essential for ensuring the effective correspondence between the real and the declared identity of a clone, in order to avoid commercial frauds and protect the legitimate interests of poplar breeders, growers and industry.

The European Union requires that propagation materials should be accompanied by a certificate of clonal identity; control and certification are demanded by nationally appointed authorities. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

The most comprehensive description system based on phenotypic traits is that adopted by UPOV (1981). 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

UPOV requires clones proposed for protection in countries adhering to the UPOV Convention, to be accompanied by a detailed description according to 64 morphological traits.

However, the only use made of the description is to help authorities in charge of the technical examination (the so-called DUS tests) to appropriately select similar varieties to be compared with the new one. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

The so-called DUS tests, for distinction, uniformity and stability do not produce a “one and for all” description of clones, but only state if the new clone can be distinguished from those already known and having similar declared features, when they have been observed simultaneously in the same environment. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

However, a control of the correspondence between the identity reported on documents and the real identity of clones is always been difficult for a number of reasons:

1. Observable traits are often affected by environmental factors.

2. Distinctive characteristics may be observable for a limited period of the year, while on site controls in nurseries can only be occasionally.

3. Individual descriptions are of limited use if two or more similar clones cannot be observed simultaneously. Control of varietal identity in the nursery chain ( controls for law)‏

poplar farmer nursery man

poplar plantlets cuttings

cutting farm nursery

poplar stand constitutor cutting farm 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

With the aim of providing certifiers with a suitable tool for the identification of one-year-old poplar clones in commercial nurseries, a dichotomous key was drawn up based on morphological traits.

This key includes 121 clones cultivated in Italy and registered in EU countries. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

Collection of clones registered in : 89 clones Other experimental and/or commercial clones: 32 Pure and hybrids among them considered clones (n.)‏ alba 1 Populus nigra 4 Populus deltoides 8 Populus trichocarpa 2 Populus ×canadensis (P. deltoides × P. 86 nigra)‏ Populus ×generosa (P. trichocarpa × P. 9 deltoides)‏ others 11 TOTAL 121 Constitution of 6 nurseries in 4 bio-geographical regions Mountain region: Aosta Continental region: Casale Monferrato (AL) and Gazzo Bigarello (MN)‏ Apennine region: Firenze and Spello (PG)‏ Mediterranean region: Roma

Experimental schemes: random blocks with 3 parcel replications each of 10 . A statistical analysis was performed.

year 2004 71 characters expressed in 676 values 3 replications 2028 values/clone 117 clones × 2 sites 474.552 total values 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

The best period to perform the survey is between the middle of August and the middle of September. In this time each R1F1 consists of a stem (1-year shoot), developed by a bud of the planted cutting, on which sylleptic branches have grown without further branching.

Both the stem and the branches bear buds - developing . morphology varies widely according to the portion where leaves are developed, the period of blooming and their age.

MORPHOLOGIC DESCRIPTION OF R1F1 POPLAR PLANTLETS The leaves of 1-year plantlets were divided in to: a) leaves of the main stem Heterophyllism b) leaves of syllpetic branches

The lamina of leaves developed by the main stem is generally more extended and its shape and trichome are often significantly different from those leaves developed by branches. The petiole is often different as well: in main stem leaves it is longer and its section is usually more or less round, whereas in branch leaves it is shorter and generally pressed and flat . Heterophyllism Besides the branch type, heterophyllism is also affected by the season when the leaf was developed.

Seasonal heterophyllism depends on the continuous leaf sprouting on shoots even after the initial blooming of the preformed embryonic leaves from winter buds.

Thus, during the vegetative season, on the same growing shoot several types of new-formed leaves are developed, placed in chronological sequence on the branch from its base towards the tip, whose morphologic features vary in connection with the physiologic changes of assimilative tissues during the ontogenetic cycle. Leaf types

In the dichotomous key, three morphometric leaf types were characterized.

FBR: spring leaves of branches (the first 5 leaves at branch base)‏

FAR: summers leaves of branches (the first 5 fully extended leaves at branch tip in the survey period)‏

FMS: most developed leaves of the main stem (appearing at the end of July / beginning of August); they are usually placed at ¾ of main stem height and are easily recognized for their widest size in the whole plant). 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

The validity of characters was evaluated taking into account their polymorphism and intraclonal variability in relation to genetic, cultural / environmental and seasonal features.

The characters selected were analyzed with a multivariate statistical approach and used in the elaboration of mathematical classification models. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

So even if in our work 202 phenological and morphological characters were analyzed in this dichotomous key 42 characters were considered, splitting them in: • main discriminants (useful for the detection of large groups)‏;

• secondary discriminants (useful for the detection of small groups or to decide between 2 clones)‏;

• complementary discriminants (useful to better describe the clone on the whole). 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

Intraclonal variability was verified according to: the different agricultural and environmental factors interacting in the nursery, like soil texture and reaction, available water and nourishing elements (introduced or not with agronomic management), the amount of light, and plant health; the physiological condition of plants (chronological variability of the character during the vegetative season or the circadian cycle), especially in connection with light and temperature; the topophysis (i.e. the new plants propagated with cuttings taken from different portions of a given plant maintain the features of the original portion); the subjectiveness of the operator in evaluating the characters. Dichotomous key for clonal identification divides the 121 clones in 10 main groups

phenotype phenotype phenotype sez. Tacamahaca Populus nigra Populus alba

phenotype Populus deltoides

phenotype “caudina”

heterogeneous group “Marilandica” group ‘I-214’ “white Canadian group ‘Dorskamp’ Lomellina” group group

23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

dichotomous keys

Deliverable descriptive cards of the single clones tables of comparison 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

Merits of the system

• applicative tool practical and easy to be used; • anatomical or hardly detectable characters are excluded from analysis; •a particular attention was given to polymorphism and intraclonal variability of all characters; • the suitability of characters was evaluated in connection with environmental, cultural and seasonal features; •morphologic characters and descriptive protocols of UPOV and IPC were considered at the same time; • the selected characters were subjected to statistical analysis. 23rd Session of the International Poplar Commission “Poplars, Willows and People's Wellbeing” Beijing, China, 26- 30 October 2008

Limitation of the system

Only suitable for plantlets at the first year of plantation, cultivated with commercial aims

Limited to the 121 considered clones

Limited period of application (September in northern Italy)‏

Lack of flower characters. MINISTERO DELLE POLITICHE AGRICOLE ALIMENTARI E FORESTALI

This dichotomous key is supplied with the CD “Dico-Pioppo”, including a PDF copy of the key, software to use with the key (assisted system) and software for unassisted clonal recognition .

Thanks for your attention !

Unità di Ricerca per le Produzioni Legnose Fuori Foresta Strada per Frassineto 35 Casale Monferrato (AL)