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e genus Pulmonaria (Boraginaceae) in the province of Bologna
PAOLO PUPILLO
Emeritus professor, Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Italy
GIANCARLO MARCONI
Associazione Pangea, San Lazzaro di Savena, Bologna, Italy
LORENZO PERUZZI
Dipartimento di Biologia, Unità di Botanica, Università di Pisa, Via Derna 1, 56126 Pisa, Italy
GIOVANNI ASTUTI
Dipartimento di Biologia, Unità di Botanica, Università di Pisa, Via Derna 1, 56126 Pisa, Italy; E-mail gastuti@biologia.unipi.it
A
Populations of Pulmonaria on Apennine highlands in the province of Bologna were investigated. Except for a few isolated sites with P. ocinalis,
most populations are referable to P. apennina on the basis of leaf shape, indumentum and chromosome number (2n = 22). Pulmonaria apennina
occurs in two morphs: i) a green morph with few or no leaf spots, common in mountain sites and similar to its Alpine vicariant, P. vallarsae; ii)
a variegated morph with diuse, clear leaf spots occurring in cool locations at low altitude, often not distant from cities. Hilly areas along the
Reno valley, however, are colonized by populations of P. hirta with 2n = 28 chromosomes, characterized by lanceolate leaf shape and extensive
maculation. Certain similarities between P. hirta and the variegated morph of P. apennina have long caused confusion and misidentications, but
the two species in the area of investigation are distinguishable on the basis of leaf morphology and indumentum.
Key words: basal leaves, foliar spots, karyology, lungwort, Pulmonaria, trichomes
R
Il genere Pulmonaria (Boraginaceae) nella Provincia di Bologna
Sono state studiate le popolazioni di Pulmonaria L. sulle montagne della provincia di Bologna. Tranne che per pochi siti isolati con P. ocinalis,
la massima parte delle popolazioni sono riferibili a P. apennina in base alla forma fogliare, all’indumento e al numero cromosomico (2n = 22).
Pulmonaria apennina si presenta in due morfe: i) una morfa verde con poche o nessuna macchia fogliare, comune in località montane e simile alla
sua vicariante delle Alpi, P. vallarsae; ii) una morfa variegata con macchie fogliari diuse, chiare, distribuita in siti freddi a bassa altitudine, spesso
non distanti dalle città. Tuttavia, le aree collinari lungo la valle del Reno sono colonizzate da popolazioni di P. hirta con 2n = 28, caratterizzate dalla
forma lanceolata delle foglie e da macchiatura abbondante. Certe somiglianze fra P. hirta e la morfa variegata di P. apennina hanno a lungo causato
confusioni e determinazioni erronee, ma le due specie nell’area in esame sono distinguibili sulla base della morfologia fogliare e dell’indumento.
Parole chiave: Foglie basali, macchie fogliari, cariologia, Pulmonaria, tricomi
I
Several species and morphs of the genus Pulmonaria L. (lun-
gworts) occur in the Bologna province, and their systematics
is still debated. e situation is clear for the European species
P. ocinalis L. (Fig. 1A), which extends its discontinuous ran-
ge in Italy as far south as the Gran Sasso massif in Abruzzo
(Bartolucci et al. 2012). All other Apennine lungworts (Figs.
1B-1E) are often thought to fall within one large species com-
plex (Lacaita 1927; Merxmüller & Grau 1969; Merxmüller &
Sauer 1972; Bolliger 1982; Tison & de Foucault 2014). An-
ton Kerner von Marilaun in his Monographia Pulmonariarum
(1878), the rst modern approach to lungwort systematics,
recognized two related Apennine species: P. vallarsae A.Kern.
and P. saccharata Mill. e former was originally described
based on plants from south-eastern Alps but later extended
to the whole of Italian peninsula as the most common Ita-
lian Pulmonaria, the latter shows strongly speckled leaves and
occurs in south-western Alps and north-central Apennines.
Although Kerner’s treatment was criticized by Lacaita (1927)
who reputed Kerner’s P. saccharata to be, in fact, a variegated
form of P. vallarsae, it was essentially accepted by all authors
of Italian Floras (Fiori 1923; Zangheri 1976; Pignatti 1982).
Puppi & Cristofolini (1991), however, argued that the name
P. saccharata is not applicable to any Italian population and
plants named P. saccharata sensu Kerner are to be referred to P.
picta Rouy. More recently, the name P. picta has been rejected
in favour of P. hirta L., the earlier name holding priority (Con-
ti et al. 2007). Puppi & Cristofolini (1996) provided evidence
that Alpine and Apennine populations attributed to P. vallarsae
are distinct enough to be separated at species level, so that the
peninsular populations were described as Pulmonaria apennina
Cristof. & Puppi, sharing with P. vallarsae the same chromo-
some number 2n = 22 (2n = 22 + 2B for specimens from Alpi
Apuane: Merxmüller & Grau 1969; Capineri 1986), although
2n = 26 has now been reported for a population of P. apennina
Quaderni del Museo Civico di Storia Naturale di Ferrara - Vol. 7 - 2019 - pp. 19-26 ISSN 2283-6918
Figure 1. Plants and basal rosette leaves of: A) Pulmonaria officinalis subsp. officinalis, Val Rio Maggiore (Sasso Marconi) June
24, 2012; B) P. vallarsae, Passo Borcola (Recoaro, Vicenza) Aug. 11, 2014; C) green morph of P. apennina, M.te Bastione (S.
Benedetto Val di Sambro) May 10, 2009; D) variegated morph of P. apennina, Dolina Spipola (S. Lazzaro di Savena) Nov.
7, 2010; E) P. hirta, Poggio di Carviano (Grizzana Morandi) Nov. 14, 2013. All from Bologna province except P. vallarsae.
(Astuti et al. 2019). On the other hand, most chromosome
counts for P. hirta gave 2n = 28, but some reports had 2n = 22
as well as intermediate numbers such as 2n = 26 and 27, thus
partially overlapping P. apennina and P. vallarsae (Merxmüller
& Grau 1969; Sauer 1975; Capineri 1986; Puppi & Cristofo-
lini 1996; Astuti et al., 2019).
In a survey of some two hundred populations of Pulmonaria
in northern Apennines, Vosa & Pistolesi (2004) found three
major karyotypes: 2n = 16 (P. ocinalis), 2n = 22 and 2n = 28
(P. apennina and/or P. hirta), with cytological hybrids between
these two species found in two sites. ese authors were unable
to correlate karyology and morphology of the plants exami-
ned and considered any distinction between P. apennina and
P. hirta as unfeasible. Consequently, some authors treated this
group of parapatric lungworts at the rank of subspecies, i.e. P.
hirta subsp. hirta and P. hirta subsp. apennina (Cristofolini &
Puppi) Peruzzi (Bernardo et al. 2010). In their recent mono-
graph of Italian Boragineae, on the other hand, Cecchi & Sel-
vi (2015) split P. vallarsae s.l. into two subspecies: P. vallarsae
subsp. apennina (Cristof. & Puppi) L.Cecchi & Selvi, and the
nominal subspecies, while no subspecies was recognized within
P P, G M, L P, G A
AB
CDE
the separated P. hirta (with 2n = 28; Cecchi & Selvi 2015). e
latter treatment has been adopted in the updated checklist of
Italian ora (Bartolucci et al. 2018).
In the attempt to clarify the situation, we have conducted a
study of Pulmonaria populations of mountain areas of the
Bologna province, which also includes the locus classicus of P.
apennina near Bologna (Peruzzi et al. 2015). Our results con-
rm the presence of three dierent chromosome numbers,
probably related to the three taxa described as P. ocinalis, P.
hirta and P. vallarsae subsp. apennina. For the sake of simplici-
ty, we shall refer to this latter taxon throughout this paper as P.
apennina, which is primarily applied to populations morpho-
logically similar to P. vallarsae and mostly featuring full-green,
uniform leaves (green morph), fairly common on mountains.
In addition, we also recognize a variegated morph related to
P. apennina, but showing some distinctive traits often inclu-
ding an extensive leaf maculation, either pale-green or white.
Pulmonaria ocinalis occurs in some isolated, cool hill sites.
Pulmonaria hirta is mainly distributed along the Reno valley
and at the border of Tuscany.
Locations: Populations of Pulmonaria were sam-
pled in the municipalities of Bologna (Colle d. Guardia
44.4791194,11.2958754; Roncrio 44.4680868,11.3348583;
Villa Ghigi 44.4767947,11.3244979), Camugnano (Ri-
gna 44.1706205,11.0838834), Casalecchio (Parco Talon
44.469414,11.2859311; Tizzano 44.4676736,11.252442),
Castel San Pietro (Monte Calderaro 44.3607437,11.485439),
Castiglione de’ Pepoli (Brasimone 44.119942,11.1158439),
Granaglione (Tre Croci 44.1136138,10.9259836; Mo-
lino del Pallone 44.0997872,10.9603472), Grizzana
Morandi (Montovolo 44.2171075,11.0846472; Pog-
gio di Carviano 44.2877256,11.1119003), Lizzano in
Belvedere (Vidiciatico 44.1699822,10.8692671), Mar-
zabotto (Medelana 44.3774143,11.1704765; Mt.
Sole 44.3155265,11.2067627), Monterenzio (road to
Monter. Vecchio 44.3231773,11.4209194), Val Sa-
moggia (Oliveto 44.4730288,11.1209472), Monzu-
no (M.te Adone 44.3402166,11.2854829; Villa Ce-
drecca 44.278493,11.261639), Porretta (Castelluccio
44.1515204,10.9251357), San Benedetto Val di Sambro
M
Figure 2. Distribution map of species and morphs of Pulmonaria in the province of Bologna: P. officinalis subsp. officinalis,
P. apennina green morph, P. apennina variegated morph, and P. hirta.
(Madonna dei Fornelli 44.2031283,11.25641; M.te Bastio-
ne 44.1594668,11.2557502), San Lazzaro di Savena (Ac-
quafredda-Spipola 44.4452735,11.3777701; Farneto-Goi-
bola 44.4375954,11.4041804), Sasso Marconi (M.te Mario
44.380054,11.2569622; Rio Sasso 44.3949208,11.2521483;
Val Rio Maggiore 44.3798475,11.2031001). A Pulmonaria
vallarsae population in beechwoods of Passo Borcola at the
border between the provinces of Trento and Vicenza (municip.
Recoaro, 1420-1600 m height) was also sampled for compa-
rison.
Cultivation: ree to ve specimens for each population were
collected in years 2013, 2014, and 2015 mostly in September
and grown in pots in the Orto Botanico of the University of
Bologna until used.
Morphological analysis: Pulmonaria species show variation in
leaf morphology such as overall shape, spot colour and outli-
ne, and hair type which are often dicult to quantify and are
subject to seasonal changes (Kerner 1878; Bolliger 1982; Astu-
ti et al. 2014). We have dened the major features of the basal
leaves using 4 easily recognizable traits for leaf shape and spot
T Pulmonaria (B) B
Pulmonaria ocinalis subsp. ocinalis.
A few, isolated populations of typical P. ocinalis are found in
very fresh broadleaf woods and chilly gorges at 300-1000 m of
elevation (Fig. 1A). Pulmonaria ocinalis of Bologna province
is easily identied by plants with a diagnostic leaf morphology
and hair types, and vivid green spots (never white) frequently
conuent (Table 2A), generally similar to their Alpine relati-
ves. e distribution of this and other Pulmonaria species and
morphs as known to us is illustrated in the map of Fig. 2.
Characterization of P. apennina.
In early stages of the present study, attention was mainly given
to Pulmonaria populations consisting of faintly spotted or un-
spotted plants (“dark”) with oval or triangular basal leaves (Fig.
R
type, with 5 states each (Astuti et al. 2014). e character-state
frequency in a population is calculated as Ns
Np *100, where Ns
is the number of individuals showing the state and Np is the
total number of individuals investigated for that population
(see Table 2). Observations were made on three young basal
rosette leaves of each individual. e abbreviations for the cha-
racter states are reported in Table 1. Hair types were examined
in fresh or desiccated material using a binocular microscope
and classied according to the hair typology and terminology
proposed by Kerner (1878) (Table 3).
Karyological analysis: Metaphase chromosome plates were
obtained by squashing root tips cells according to the fol-
lowing protocol: pretreatment in 0.4% colchicine solution for
3 hr; Carnoy xing for one hr; hydrolysis in HCl 1N for 9 min
at 60°C; staining with leucobasic fucsine for at least 2 hr.
Spot type
Apparently no spots Very small Roundish, regularly
scattered
Shape and distribution
irregular (mortar-like)
Spots widely conuent
ns sm ro mo cf
Spot colour
Ground green Inconspicuous
(but visible)
Gaudy green Light green
to yellowish
Bold white
iv ic gr ye bw
Leaf base
Cordate (indented)
with peduncle
Truncate with
distinct peduncle
Arcuate
(crescent-shaped)
with peduncle
Shortly decurrent
on peduncle
Tapering into
winged peduncle
cd tr ar dr wg
Leaf shape
Roundish (plump) Heart-shaped Oval to spear-shaped Elongated* Very long & thin
pl he sp el th
Table 1. Major traits (character-states) of Pulmonaria basal leaves used as combined population markers. Abbreviations in
bold. Elongated*: about 2× longer than wide at max leaf width.
1C). Such plants are fairly common in faraway mountain loca-
lities, but also in fresh woods and slopes near Bologna. A sta-
tistical prole of the morphological traits of a “dark” lungwort
population, reported in Table 2C, supports the assignment to
P. apennina and remarks an overall resemblance to P. vallarsae
(Fig. 1B and Table 2B). e diagnostic soft hair patterns with
abundant short hairs (puberes) and some bristles (setae) t the
protologue of P. apennina (Table 3). We refer to these wide-
spread P. apennina plants (Fig. 2) as the green morph.
Several populations of Pulmonaria also referable to P. apenni-
na, but showing partially divergent morphological traits, are
found in cool, shady sites often not distant from human settle-
ments (Fig. 2). An interesting example is the lungwort belt on
hills overlooking Bologna, in part on gypsum rocks (Parco dei
Gessi Bolognesi). ese plants are clearly related to P. apennina
on the basis of hair types and karyology (see below), but they
often display large, irregular leaf spots or even spot conuence
particularly in autumn (Fig. 1D, Table 2D), which can result
in a certain resemblance to P. hirta. We refer to this type as the
variegated morph of P. apennina.
Characterization of P. hirta.
Typical features of this plant group are elongated summer le-
aves showing setae and few puberes (contrary to P. apennina),
and a gaudy leaf speckling ranging from pale green to bold
white, which often changes to a conuent, “glazed” leaf surface
in autumn (Fig. 1E). However, some specimens with limited
maculation also occur. A statistical analysis of P. hirta morpho-
logy reveals signicant dierences with respect to the variega-
ted morph of P. apennina, in particular longer and slenderer
leaves (lanceolate) in all seasons (Table 2E, Table 3). Plants
showing these characters are infrequent around Bologna, whe-
re the variegated morph of P. apennina is dominant, but they
are widely distributed on hills on the right hand of river Reno
P P, G M, L P, G A
T Pulmonaria (B) B
Table 2A - Pulmonaria officinalis
Val Rio Maggiore (Sasso Marconi), 94 plants - Oct. 12, 2011
Spot color Spot type Leaf base Leaf shape
ns 2 2% iv 1 1% cd 18 19% pl 4 4%-
ic 24 26% sm 1 1% tr 46 49% he 52 55%
gr 67 72% ro 28 30% ar 28 30% sp 32 34%
ye 4 4% mo 56 60% dr 1 1% el 6 6%
bw - -cf 8 8% wg - -th - -
TYPE: gr 72% - mo 60% - tr 49% - he 55%
Table 2B - Pulmonaria vallarsae
Passo Borcola, 126 plants - Aug. 11, 2014
Spot color Spot type Leaf base Leaf shape
ns 35 28% iv 28 22% cd 11 9% pl 7 6%-
ic 66 52% sm 41 33% tr 78 63% he 84 67%
gr 25 20% ro 54 43% ar 33 27% sp 32 25%
ye - -mo 3 2% dr 1 1% el 3 2%
bw - -cf - -wg - -th - -
TYPE: ic 52% - ro 43% - tr 63% - he 67%
Table 2C - Pulmonaria apennina green morph
Poggio dell’Oca, 42 plants - June 2, 2011
Spot color Spot type Leaf base Leaf shape
ns 22 52% iv 22 52% cd 5 12% pl 1 2%-
ic 10 24% sm 13 31% tr 24 57% he 21 50%
gr 5 12% ro 7 17% ar 12 29% sp 14 33%
ye 5 12% mo - -dr 1 2% el 6 14%
bw - -cf - -wg - -th - -
TYPE: ns 52% - iv 52% - tr 57% - he 50%
Table 2D - Pulmonaria apennina variegated morph
Valle dell’Acquafredda, 442 plants - July 12, 2013
Spot color Spot type Leaf base Leaf shape
ns 5 1% iv 4 1% cd 16 4% pl 5 1%
ic 16 4% sm 6 1% tr 146 33% he 182 41%
gr 101 23% ro 37 8% ar 238 55% sp 140 32%
ye 161 36% mo 314 71% dr 37 8% el 109 25%
bw 159 36% cf 81 18% wg - -th 6 1%
TYPE: ye/bw 72% - mo 71% - ar 55% - he 41%
Table 2E - Pulmonaria hirta
Poggio di Carviano, 51 plants - July 12, 2008
Spot color Spot type Leaf base Leaf shape
ns 1 2% iv 1 2% cd - -pl - -
ic 4 8% sm 4 8% tr - -he - -
gr 32 63% ro 13 25% ar 24 47% sp 19 37%
ye 11 22% mo 28 55% dr 25 49% el 8 16%
bw 3 6%- cf 5 10% wg 2 4%- th 24 47%
TYPE: gr 63% - mo 55% - dr 49% - th 47%
Table 2. A comparison of character-states of basal leaves of five lungwort populations belonging to: A) Pulmonaria officinalis
subsp. officinalis; B) P. vallarsae; C) green morph of P. apennina; D) variegated morph of P. apennina; E) P. hirta. Except for
P. vallarsae of Passo Borcola (Vicenza), all populations examined grow within the Bologna province as indicated.
According to our ndings, lungworts in the Bologna province
can be assigned to three groups. A rst group is represented by
scattered populations of P. ocinalis subsp. ocinalis occurring
in cool, shady mountain woods and brook gorges, probably re-
presenting a relict of colder climates of the past. An instructive
example is given by a healthy P. ocinalis population in the
inner, sheltered part of Val Rio Maggiore near Sasso Marconi,
where P. apennina (green morph) occupies the lower, open
part of the small valley. ese plants have a distinctive mor-
phology typical of P. ocinalis subsp. ocinalis, in contrast to
the southern Alps where, in addition to the diuse nominal
subspecies, high-mountain P. ocinalis subsp. marzolae and
other morphs also occur (Astuti et al. 2014). Pulmonaria o-
cinalis is unequivocally characterized by 2n = 16 chromosomes
(Astuti et al. 2014, and literature therein).
A second group of lungworts consists of populations of P. hir-
ta, mainly distributed along the valley of river Reno, where it
seems to largely replace P. apennina. e map of Fig. 2 shows
the dierent, major areas of occurrence of this and other Pul-
monaria species and morphs in Bologna province as known to
us, although other lungwort populations undoubtedly exist.
e Reno valley was used for thousands of years as a major
route to Tuscany and central Italy and it cannot be excluded
that the present distribution of P. hirta in the area was inuen-
ced by human activities; in addition the Montovolo–Vigese
D
in chestnut groves and along roads and ditches at 400-700 m
elevation, mainly in the municipalities of Grizzana and Camugna-
no (Fig. 2).
Karyology.
Several Pulmonaria populations mentioned in this paper were
sampled and metaphase plates of one or more plants were in-
spected. All plants labelled as the green morph of P. apennina
had a chromosome number 2n = 22 (Fig. 3A). A limited sam-
ple of the variegated morph also showed 2n = 22 (Fig. 3B).
All plants identied as P. hirta on the basis of general mor-
phology and hair types had a chromosome number 2n = 28
(Fig. 3C). No plants with intermediate chromosome numbers
were found. In our area of investigation, therefore, leaf and
hair morphology appear to be related to chromosome set.
Table 3. Pulmonaria trichomes. Comparing the hair types of the green and variegated morphs of P. apennina with those of P.
hirta. Kerner’s (1878) hair types in brackets.
Summer basal leaves,
upper face
P. apennina green morph P. apennina variegated morph P. hirta
Bristles (“Setae”) sparse sparse sparse
Short hairs (“Puberes”) numerous numerous scanty
Small pins (“Aculeoli”) absent absent absent
Glandular hairs (“Colleteres”) present present present
Microglands (sessile glands) sparse sparse sparse
range, dominating the high course of river Reno, has been an
important religious area since Etruscan times. Most of these
plants show elongated summer leaves with conspicuous (“mor-
tar-like”) to conuent spots, and a strigose indumentum for-
med by bristles (setae) and a few short hairs (puberes) (Puppi
& Cristofolini 1996). All P. hirta plants sampled had 2n = 28
chromosomes. is species has a mainly western distribution
in Italy in continuity with southern France, and a southern
limit at the latitude of Rome as stated by Vosa & Pistolesi
(2004) for their 2n = 28 cytotype. Puppi & Cristofolini (1996)
reported on the presence of P. hirta (under the name P. picta
Rouy) in high woods up to the tree line (e.g., at Lago Baccio,
Modena, with 2n = 22 chromosomes). Again, this point needs
conrmation because of possible confusion with the variegated
morph of P. apennina.
e third and more diverse group is represented by the wi-
despread and dominant P. apennina, which is found in many
localities of the area considered (Fig. 2). Most mountain
populations of P. apennina are faintly spotted or unspotted
(green morph) and resemble the south-east-Alpine P. vallar-
sae in many details, notably the chromosome number 2n =
22. Plants corresponding to the description of P. apennina are
common in secluded localities e.g., along northern slopes of
the Contraorte Pliocenico Reserve, but also in fresh hillsi-
des around Bologna as already known to Bertoloni (1855) and
Kerner (1878, p. 19: “Jola, Ronc(o)rio, alle Grotte”). At least
two-thirds of the P. apennina locations known to us in the Bo-
logna province (and in adjoining Romagna mountains to the
south-east, down to Marche: D. Ubaldi, pers. comm.) pertain
Figure 3. Examples of mitotic metaphase plates: A) of P. apennina green morph from M.te Adone (Monzuno), B) of P. apennina
variegated morph from Valle dell’Acquafredda (S. Lazzaro di Savena), C) plate of P. hirta from Poggio di Carviano (Grizzana).
AB C
to the green morph, and the original circumscription of this
species as found in central and southern Apennines also refers
to weakly spotted or unspotted plants (Puppi & Cristofolini
1996). e close relationship between P. apennina and P. val-
larsae and the wide highland distribution of the green morph
suggest that the latter may be the original, dominant form of
Apennines. It can be supposed that there was once a continu-
ity of this species from southern Alps to Apennines across the
Po plain in postglacial times, although only a few traces of it,
if any, remain by now in the Pianura Padana. On the other
hand, the variegated morph may be the product of occasional
gene ow between P. hirta and the green morph of P. apennina.
e two Apennine taxa P. apennina and P. hirta are reported to
be interfertile (Puppi & Cristofolini 1996), as also witnessed
by the occurrence of plants with chromosome numbers inter-
mediate between 2n = 22 and 2n = 28 (Puppi & Cristofolini
1996; Vosa & Pistolesi 2004; Astuti et al. 2019). In addition,
recent phylogenetic and DNA ngerprint analyses (Liu et al.,
unpublished) have conrmed that some interspecic gene ow
does occur between P. apennina (including that from the locus
classicus in the vicinity of Bologna) and P. hirta.
While the green morph of P. apennina dominates the moun-
tains, other populations of P. apennina are from moderately to
heavily spotted and can even show conuent speckling (varie-
gated morph), to the point that some individuals may resemble
P. hirta at a rst glance (Fig. 1D-E). is deceiving resemblan-
ce may be at the origin of reports of 2n = 22 chromosome
numbers for P. hirta (as P. saccharata or P. picta: Merxmüller
& Grau 1969; Sauer 1975; Puppi & Cristofolini 1996), con-
P P, G M, L P, G A
e collaboration of Lijuan Liu, Michele Bresadola, and Fran-
cesca Lentini in parts of this work is acknowledged. We also
wish to thank the sta of the Orto Botanico of Bologna Uni-
versity: C. Bonglioli, M. Bartolini, L. Magagnoli, and the
curator Umberto Mossetti for helpful assistance.
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tributing to a rather confusing situation until today. Kerner
(1878) himself considered plants from Bologna as P. sacchara-
ta, although no P. hirta is present in the outskirts of Bologna,
where the variegated morph of P. apennina is still abundant in
some shady, cool places (karst valleys in gypsum of the Acqua-
fredda-Spipola area, in particular) and along hill roads. Despite
their variable macromorphological characters, all P. apennina
populations can be recognized by a set of specic traits: trian-
gular or oval leaf form, soft indumentum with plentiful short
hairs, and a diploid chromosome number 2n = 22. e variable
appearance of variegated plants undoubtedly contributed to
the fame of morphological instability surrounding the genus
Pulmonaria, at least in Italy (Lacaita 1927; Pignatti 1982; Vosa
& Pistolesi 2004).
Our ndings, therefore, agree with Vosa & Pistolesi (2004)
that all properly examined Pulmonaria of northern and central
Apennines fall within three sharply distinct karyotype classes,
with 2n = 16 (P. ocinalis), 2n = 22 and 2n = 28 (P. hirta
complex). However, we observe that the 2n = 22 karyotype is
mostly correlated with microcharacters typical of P. apennina,
both in its green and variegated morphs, whereas 2n = 28 is
related to microcharacters typical of P. hirta. Cecchi & Selvi
(2015) also refer to P. hirta as having 2n = 28 chromosomes. In
general, we nd that the distinctive characters of P. hirta with
respect to the variegated morph of P. apennina mainly rely on
the relative abundance of short hairs (more puberes in the lat-
ter) (Table 3), in accordance with Puppi & Cristofolini (1996),
and on the dierences in leaf shape (Table 2D-E).
Current evidence supports a simple scheme with P. apennina
and the closely related P. vallarsae having 2n = 22, and P. hir-
ta having 2n = 28 chromosomes. is could help clarifying
the systematic position of this species complex. However, the
variability of variegation patterns in P. apennina, and in P. hir-
ta to some extent, is not easy to understand. at it origina-
ted by a mechanism of hybridization was early proposed by
Lacaita (1927). Vosa & Pistolesi (2004) reported that only a
few hybrids were found in their extensive investigations, but
Astuti et al. (2019) found that all individuals sampled from a
population referable to P. apennina in Abruzzo had invariably
2n = 26 (see also Capineri 1986). It may be hypothesized that
Pulmonaria hybrids have a lower tness, which would account
for their rarity in nature as already pointed out by Meeus et al.
(2016). e relationships of the two morphs of P. apennina,
green and variegated one, therefore deserve to be investigated
in terms of possible genetic introgression between the inter-
fertile (Puppi & Cristofolini 1996) and partially sympatric P.
apennina and P. hirta, although a more complex scenario of
the origins of Pulmonaria populations in northern Apennines
cannot be ruled out at this stage of knowledge.
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