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Towards a better understanding of polyploid Sorbus (Rosaceae) from Bosnia and Herzegovina (Balkan Peninsula), including description of a novel, tetraploid apomictic species: Polyploid Sorbus From Bosnia and Herzegovina

June 2015
Botanical Journal of the Linnean Society 178(4)

June 2015
178(4)

DOI:10.1111/boj.12289

Authors:

Alma Hajrudinović-Bogunić

University of Sarajevo

Božo Frajman

University of Innsbruck

Peter Schönswetter

University of Innsbruck

Elma Silajdzic

Show all 6 authorsHide

Sorbus subgenus Soraria encompasses taxa originating from spontaneous hybridization between members of subgenera Aria and Sorbus disjunctly distributed across Europe and Asia. Using molecular data (amplified fragment length polymorphisms, plastid DNA sequences and nuclear microsatellites), flow cytometry (allowing for the determination of ploidy and mode of reproduction) and morphology, we disentangled the relationships among polyploid cytotypes and explored their relationships with their diploid ancestors. Among others, we focused on a large, geographically isolated hybrid population in Bosnia and Herzegovina. Molecular and morphological analyses confirmed the distinct position of this population in relation to its parental (S. aria and S. aucuparia) and other hybridogenous taxa originating independently from the same parents in different parts of Europe. After establishing its genetic and morphological divergence, we describe the isolated Bosnian population as a new tetraploid apomictic species, Sorbus bosniaca, discuss its taxonomic status and propose conservation measures to protect the locus classicus of this new Balkan endemic. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00, 000–000.

Geographical origin and amplified fragment length polymorphism (AFLP) and plastid DNA variation of analysed Sorbus accessions. A, NeighborNet diagram of AFLP data supplemented with bootstrap values > 90% derived from a neighbour-joining analysis given for major groups only. Symbols used for species and cytotypes are the same as in (B), (C) and (D). B, Statistical parsimony networks of plastid trnT-trnF haplotypes. Sizes of circles reflect haplotype frequencies. As no intrapopulational variation was encountered, only one sequence per population was included. Symbols indicate the occurrence of a haplotype in a taxon. Numbers correspond to sampling sites in Table 1. Small black dots represent unsampled haplotypes. C–D, Localities of sampling sites (numbered 1–12; Table 1) in Europe (C) and in Bosnia and Herzegovina (D).

…

. Inferred ploidy and averaged descriptive statistics of holoploid (2C) and monoploid (1Cx) genome size values estimated for the analysed Sorbus taxa or cytotypes

…

Illustration of Sorbus bosniaca. A, Flowering long shoot bearing a lateral sterile short shoot. B, Flower in longitudinal section. C, Flower from above. D, Ripe pome.

…

Holotype of Sorbus bosniaca.

…

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Towards a better understanding of polyploid Sorbus

(Rosaceae) from Bosnia and Herzegovina (Balkan

Peninsula), including description of a novel, tetraploid

apomictic species

ALMA HAJRUDINOVIC

´1, BOŽO FRAJMAN2, PETER SCHÖNSWETTER2,

ELMA SILAJDŽIC

´1, SONJA SILJAK-YAKOVLEV3and FARUK BOGUNIC

´1*

1Faculty of Forestry, University of Sarajevo, Zagrebacˇka 20, 71000 Sarajevo, Bosnia and Herzegovina

2Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria

3CNRS, Université Paris-Sud, AgroParisTech, UMR 8079, Ecologie, Systématique, Evolution, Bât.

360, 91450 Orsay, France

Received 17 December 2014; revised 20 February 2015; accepted for publication 6 April 2015

Sorbus subgenus Soraria encompasses taxa originating from spontaneous hybridization between members of

subgenera Aria and Sorbus disjunctly distributed across Europe and Asia. Using molecular data (ampliﬁed

fragment length polymorphisms, plastid DNA sequences and nuclear microsatellites), ﬂow cytometry (allowing for

the determination of ploidy and mode of reproduction) and morphology, we disentangled the relationships among

polyploid cytotypes and explored their relationships with their diploid ancestors. Among others, we focused on a

large, geographically isolated hybrid population in Bosnia and Herzegovina. Molecular and morphological analyses

conﬁrmed the distinct position of this population in relation to its parental (S. aria and S. aucuparia) and other

hybridogenous taxa originating independently from the same parents in different parts of Europe. After estab-

lishing its genetic and morphological divergence, we describe the isolated Bosnian population as a new tetraploid

apomictic species, Sorbus bosniaca, discuss its taxonomic status and propose conservation measures to protect

Linnean Society, 2015, 00, 000–000.

ADDITIONAL KEYWORDS: apomixis – genome size – hybridization – morphology – polyploidy.

INTRODUCTION

Interspeciﬁc hybridization accompanied by polyploidi-

zation (allopolyploid hybridization) is a powerful

factor driving and shaping plant diversiﬁcation

(Rieseberg & Willis, 2007; Hegarty & Hiscock, 2008;

Soltis & Soltis, 2009). Allopolyploid genome multipli-

cation may profoundly affect genome structure and

gene expression and also induce morphological

changes (Soltis & Soltis, 2009; Balao, Herrera &

Talavera, 2011; D

ˇurkovicˇ et al., 2012). In addition,

hybridization and polyploidy may be followed by a

change in the reproductive mode, usually from sexual

to asexual reproduction (apomixis, agamospermy;

Hörandl, 2006). On the one hand, this transition

leads to reproductive isolation of newly formed off-

spring from the parents and, on the other, it enables

the establishment and maintenance of viable popula-

tions via facultative or obligate apomixis (Ludwig

et al., 2013). Although obligate apomicts do not repro-

duce sexually, they may undergo regular microsporo-

genesis and yield fertile pollen, thus being able to

participate in further gene exchange (Robertson et al.,

2010). The diversity generated by the recurrent inter-

action of hybridization, polyploidy and apomixis

renders several species groups a nightmare for tax-

onomists, a prime example being the European rep-

resentatives of the genus Sorbus L.

*Corresponding author. E-mail: faruk.bogunic@gmail.com

bs_bs_banner

Botanical Journal of the Linnean Society, 2015, ••, ••–••. With 4 ﬁgures

Sorbus (Rosaceae) comprises >250 species inhabit-

ing the Northern Hemisphere (Phipps et al., 1990).

The centre of diversity of the genus is East Asia

(Aldasoro et al., 2004); Europe is considered as a

secondary diversity centre, with three hotspots of

ongoing diversiﬁcation and speciation located in Scan-

dinavia, south-eastern Europe and Britain (Jankun,

1993; Warburg & Kárpáti, 1993; Rich et al., 2010;

Robertson et al., 2010). It has been hypothesized that

diversiﬁcation of European Sorbus has initially been

driven by hybridization and backcrosses of the four

widely distributed parental diploids S. aria (L.)

Crantz s.s.,S. aucuparia L., S. chamaemespilus (L.)

Crantz and S. torminalis (L.) Crantz (Warburg &

Kárpáti, 1993; Rich et al., 2010; Robertson et al.,

2010). Most hybrid derivatives are apomictic tri- and

tetraploids restricted to geographical areas of varying

size (Lepší et al., 2008, 2009, 2013; Rich et al., 2010;

Robertson et al., 2010). Flora Europaea (Warburg &

Kárpáti, 1993) recognizes 91 Sorbus spp. and 15 sub-

species. Full descriptions are only provided for 18 of

these, whereas the others are only mentioned as

‘closely related species’. This number is increasing

steadily (Rich & Houston, 2006; Robertson & Sydes,

2006; Lepší et al., 2008, 2009, 2013; Castellano et al.,

2012; Raimondo et al., 2012) and, in the Euro + Med

Plantbase, 157 Sorbus spp. are listed (Kurtto, 2009).

At the other extreme, some authors recognize only 12

species in Europe (Aldasoro et al., 1998), reﬂecting

the application of different species concepts.

The Balkan Peninsula, one of the major European

diversity hotspots (Kryštufek & Reed, 2004; Frajman,

Pachschwöll & Schönswetter, 2014), hosts c. 30 taxa,

including 18 species and 12 subspecies of Sorbus

(Beck-Mannagetta, 1927; Jovanovic´, 1972; Polunin,

1987; Nikolic´, 1997; Micevski, 1998; Delipavlov et al.,

2003; Pulevic´, 2005; Martincˇicˇet al., 2007), account-

ing for almost one-third of European Sorbus diversity

(Warburg & Kárpáti, 1993). Nevertheless, knowledge

is far from complete and, for instance, populations

belonging to the S. latifolia (Lam.) Pers. group were

only recently discovered in Bosnia and Herzegovina

(Hajrudinovic´, Bašic´ & Bogunic´, 2012).

Sorbus subgenus Soraria Májovský & Bernátová

comprises di-, tri- and tetraploid taxa, which origi-

nated from hybridization and backcrosses between

members of subgenera Aria Pers. and Sorbus

(Májovský & Bernátová, 2001). Although seven taxa

have been reported for the Balkan Peninsula

(Beck-Mannagetta, 1927; Jovanovic´, 1972; Nikolic´,

1997; Micevski, 1998; Delipavlov et al., 2003; Pulevic´,

2005; Martincˇicˇet al., 2007), only S. austriaca

(Beck) Hedl. and S. ×thuringiaca (Nyman) Schönach

(= S. ×semipinnata Hedl.) have been reported from

Bosnia and Herzegovina, the latter from three locali-

ties (Beck-Mannagetta, 1927). However, although mor-

phological inspection conﬁrmed the assignment to S.

subgenus Soraria, preliminary ﬂow cytometric data

(A. Hajrudinovic´, F. Bogunic´, S. Siljak-Yakovlev,

unpubl. data) of plants from one locality (Mt. Krug

planina) revealed that this population is tetraploid,

whereas typical S. ×thuringiaca is diploid (Rich et al.,

2010).Overall, more than 300 adult fructiferous indi-

viduals occurring in sympatry with other Sorbus spp.

were counted on Mt. Krug planina (A. Hajrudinovic´

and F. Bogunic´, unpubl. data). The other two localities

could either not be traced (Mt. Maglic´) or are inacces-

sible because of mine ﬁelds (Koric´ino sedlo).

Here, our main aim was to disentangle the rela-

tionships among polyploid cytotypes of Sorbus subge-

nus Soraria from Bosnia and Herzegovina, to explore

their relationships with similar taxa from other parts

of Europe and to elucidate their origin from diploid

ancestors. We employed an array of molecular tech-

niques, i.e. ampliﬁed fragment length polymorphism

(AFLP) ﬁngerprinting, plastid DNA sequencing,

nuclear microsatellites and rbcS genotyping. Flow

cytometry was used for the determination of genome

size and ploidy and to determine the mode of repro-

duction via seed screening. As the genetic and cyto-

logical data supported the tetraploid population from

Mt. Krug planina in western Bosnia as an independ-

ent evolutionary lineage, morphological characters

were evaluated as a basis for a formal description of

the new species.

MATERIAL AND METHODS

PLANT MATERIAL

Leaf material was collected in the ﬁeld from sampling

sites 1–5 (Table 1, Fig. 1) and dried in silica gel for

molecular studies, and fresh leaves were used for ﬂow

cytometric analysis. From sampling site 6, only

winter buds were available. Voucher numbers and the

location of vouchers are given in Table 1. Leaf

samples of Northern European populations of diploid

S. aria,S. aucuparia,S. hybrida L. and S. × thuringi-

aca (sampling sites 7–11) were provided by M. Fay

(Jodrell Laboratory, Royal Botanic Gardens, Kew,

UK) and leaf material of S. borbasii Jáv. from the

locus classicus was provided by C. Németh (Depart-

ment of Botany, Corvinus University, Budapest,

Hungary). Seeds of S. bosniaca were collected for the

ﬂow cytometric seed screen.

FLOW CYTOMETRY

The total nuclear DNA content was estimated for 151

individuals, following Marie & Brown (1993)

(Table 2). Genome size estimates for seven individu-

als from England, Finland and a single individual

from Austria (sampling sites 7–11; Table 1) were

2A. HAJRUDINOVIC

´ET AL.

Table 1. Geographical origin and sample sizes for ampliﬁed fragment length polymorphism (AFLP) and nuclear microsatellite analyses (samples for plastid DNA

sequencing in parentheses) of analysed Sorbus accessions

Sampling site

S. aria

S. aucuparia

S. austriaca

S. borbasii

S. bosniaca

S. hybrida

S. ×thuringiaca

2xTotal

1Gornja Grkarica, Mt. Igman, Bosnia and

Herzegovina, 43°43′05″N, 18°18′22″E, 1300 m;

Leg. A. Hajrudinovic´, F. Bogunic´, SARA (51415;

51416; 51417; 51418)

6 (3) 3 (3) 10 (3) 10 (3) 29 (12)

2Umoljani, Mt. Bjelašnica, Bosnia and

Herzegovina, 43°39′31″N, 18°13′49″E, 1260 m;

Leg. A. Hajrudinovic´, F. Bogunic´, SARA (51412;

51413; 51414)

6 (3) 9 (2) 6 (3) 21 (8)

3Klinje lake, Bosnia and Herzegovina,

43°10′55″N, 18°34′02″E, 1090 m; Leg. A.

Hajrudinovic´, F. Bogunic´, SARA (51411)

9 (2) 9 (2)

4Mt. Krug planina, Bosnia and Herzegovina,

43°50′32″N, 17°11′59″E, 1300 m; Leg. A.

Hajrudinovic´, F. Bogunic´, SARA (51406; 51407;

51408; 51409), WU (080424)

14 (3) 4 (3) 13 (2) 2 (2) 20 (5) 53 (15)

5Mt. Slovinj, Bosnia and Herzegovina,

43°59′41″N, 16°59′06″E, 1350 m; Leg. A.

Hajrudinovic´, F. Bogunic´, SARA (51410)

8 (3) 8 (3)

6Innsbruck, Tirol, Austria, 47°16′37″N,

11°22′35″E, 900 m; Leg. P. Schönswetter, B.

Frajman, A. Hajrudinovic´, no vouchers

7 (3)* 9 (3)* 16 (6)

7Hainburg, Niederösterreich, Austria (FC382

from Pellicer et al., 2012)

1 (1) 1 (1)

8The Plain, Leigh Woods, England, UK (FC007,

FC001 from Pellicer et al., 2012)

1 (1) 1 (1) 2 (2)

9Offa’s Dyke, Tidenham Chase, Wales,

UK (FC154 from Pellicer et al., 2012)

1 (1) 1 (1)

10 Åland, Lemland, Finland; (FC375, FC376 from

Pellicer et al., 2012)

2 (2) 2 (2)

11 Uusimaa, Vihti, Finland; (FC369, FC370 from

Pellicer et al., 2012)

2 (2) 2 (2)

12 Mt. Domugled, Herkülesfürdo˝ , Romania; Leg.

D. Pifkó, Cs. Németh, B. Pintér, BP (702342)

3† (–)

Total 31 (11) 16 (9) 12 (5) 35 (11) 27 (10) 3 (–) 20 (5) 2 (2) 1 (1) 144/147†(54)

*Ploidy inferred from nuclear microsatellites based on the maximal number of displayed alleles per analysed locus.

†S. borbasii was included only in the microsatellite dataset. The sequence of voucher numbers reﬂects the order of taxa or cytotypes in the table.

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 3

S. aria (2x)

S. aria (3x)

S. aria (4x)

S. austriaca (4x)

S. bosniaca (4x)

S. hybrida (4x)

S. aucuparia (2x)

S. x thuringiaca (2x)

S. borbasii (4x)

0.01

100

Sorbus aria

haplotype group

Sorbus aucuparia

haplotype group

4 1, 2, 3, 4, 6, 8, 10, 11 61

24 1, 2, 4, 5, 6, 7

Figure 1. Geographical origin and ampliﬁed fragment length polymorphism (AFLP) and plastid DNA variation of

analysed Sorbus accessions. A, NeighborNet diagram of AFLP data supplemented with bootstrap values >90% derived

from a neighbour-joining analysis given for major groups only. Symbols used for species and cytotypes are the same as

in (B), (C) and (D). B, Statistical parsimony networks of plastid trnT-trnF haplotypes. Sizes of circles reﬂect haplotype

frequencies. As no intrapopulational variation was encountered, only one sequence per population was included. Symbols

indicate the occurrence of a haplotype in a taxon. Numbers correspond to sampling sites in Table 1. Small black dots

represent unsampled haplotypes. C–D, Localities of sampling sites (numbered 1–12; Table 1) in Europe (C) and in Bosnia

and Herzegovina (D).

4A. HAJRUDINOVIC

´ET AL.

taken from Pellicer et al. (2012). Fresh Sorbus

leaves (c.1cm

2) were co-chopped with leaves of

internal standards [Solanum lycopersicum L. ‘Roma’,

2C = 1.99 pg (Marie & Brown, 1993) or Salvia

brachyodon Vandas, 2C = 0.95 pg (Maksimovic´ et al.,

2007)] in 600 μL cold Galbraith’s isolation buffer

(Galbraith et al., 1983) supplemented with 1% poly-

vinylpyrrolidone (∼10 000 Mr, Sigma) and 5 mM

sodium metabisulphite (Roche). The suspension was

ﬁltered through a 50-μm CellTrics mesh (Partec) and

2.5UmL

−1 RNase A (Roche) was added. Nuclei were

stained with propidium iodide (Sigma-Aldrich) at a

ﬁnal concentration of 50 μgμL−1. The solution was

incubated on ice for 20–30 min prior to analysis. The

ﬂuorescence of 5000 nuclei was recorded using a

Partec CyFlow SL3 532-nm laser cytometer (Partec).

Fluorescence histograms were analysed in FloMax

ver. 2.6 (Partec). The holoploid genome size values

(2C; Greilhuber et al., 2005) were calculated using the

linear relationship between the ﬂuorescence of

stained nuclei of the sample and the internal stand-

ard of known genome size. DNA ploidy (Suda et al.,

2006) was inferred using earlier chromosome counts

of Sorbus spp. done by Siljak-Yakovlev et al. (2010),

and monoploid values (1Cx, Greilhuber et al., 2005)

were calculated.

FLOW CYTOMETRIC SEED SCREEN AND

POLLEN VIABILITY

Flow cytometry was used for the determination of

reproductive mode following Matzk, Meister &

Schubert (2000). Seeds were kept at 4 °C for about 7

months before analysis. The protocol was identical to

that described above except for the use of Oryza sativa

L. ssp. japonica ‘Nipponbarre’ (2C = 0.9 pg; Uozu et al.,

1997) as the internal standard. Thirty-four seeds were

analysed separately. The DNA amount of the embryo

was used to infer the monoploid genome size needed for

the calculation of endosperm ploidy. Estimated DNA

ploidies of embryo and endosperm were compared to

distinguish between sexual reproduction and apomixis

(Supporting Information, Appendix S1). The determi-

nation of reproduction mode and the interpretation of

the pathway of endosperm formation followed previous

reports (Talent & Dickinson, 2007a, b; Cosendai &

Hörandl, 2010; Šarhanová et al., 2012) on plant groups

having the same Polygonum-type of embryo sac as

Sorbus (Liljefors, 1953).

Pollen viability was tested for 20 S. bosniaca indi-

viduals using Alexander’s reagent (Alexander, 1969).

Fresh pollen grains from open anthers were placed on

a microscopic slide in a drop of Alexander’s stain and

left for 24 h at room temperature. One hundred

grains were counted per individual and viability was

expressed as a percentage. Well-formed grains with

green-stained cell walls and homogeneously bright

red cytoplasm were considered as viable.

AFLP

One hundred and forty-four individuals were

included (Table 1). A modiﬁed cetyltrimethylammo-

nium bromide (CTAB) procedure (Tel-Zur et al., 1999)

was used for the extraction of total genomic DNA from

c. 20 mg of silica-dried leaf material. The AFLP proto-

col followed Vos et al. (1995) with modiﬁcations

described in Schönswetter et al. (2009). In addition,

0.25 U Red Taq polymerase (Sigma-Aldrich) was

used for preselective ampliﬁcation and selective

Table 2. Inferred ploidy and averaged descriptive statistics of holoploid (2C) and monoploid (1Cx) genome size values

estimated for the analysed Sorbus taxa or cytotypes

Taxon

DNA

ploidy N

2C DNA (pg) 1Cx DNA (pg)

Mean ± SD Min–Max CV (%) Mean ± SD Min–Max

S. aria 2x35 (2*) 1.38 ± 0.04 1.30–1.49 2.92 0.69 ± 0.02 0.65–0.74

S. aria 3x16 2.01 ± 0.07 1.86–2.10 3.29 0.67 ± 0.02 0.62–0.70

S. aria 4x12 2.68 ± 0.07 2.57–2.76 2.45 0.67 ± 0.02 0.64–0.69

S. aucuparia 2x20 (3*) 1.39 ± 0.06 1.31–1.53 4.19 0.69 ± 0.03 0.65–0.76

S. austriaca 4x26 2.69 ± 0.07 2.52–2.78 2.72 0.67 ± 0.02 0.63–0.70

S. bosniaca 4x42 2.69 ± 0.05 2.60–2.83 1.88 0.67 ± 0.01 0.65–0.71

S. hybrida 4x2* 2.95 ± 0.05 2.91–2.98 1.82 0.74 ± 0.01 0.73–0.75

S. ×thuringiaca 2x1* 1.51 ± 0.00 1.51–1.51 0.00 0.75 ± 0.00 0.75–0.75

Total 151 (8*)

CV, coefficient of variation; Min, minimum; Max, maximum; SD, standard deviation.

*Genome sizetaken from Pellicer et al. (2012).

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 5

ampliﬁcations with 6-FAM and VIC-labelled primers,

whereas 0.4 U was used with the NED-labelled primer

(6-FAM-labelled primers, Sigma-Aldrich; NED- and

VIC-labelled primers, Applied Biosystems). Twelve

selective primer combinations were initially screened.

The ﬁnal three selective primer combinations for the

selective polymerase chain reaction (PCR) (ﬂuorescent

dye in parentheses) were EcoRI (6-FAM)-ACA/

MseI-CAC, EcoRI (VIC)-AAG/MseI-CTG, and EcoRI

(NED)-ACC/MseI-CAG (MseI and EcoRI primers,

Sigma-Aldrich). Puriﬁcation and visualization of PCR

products were performed as described in Rebernig

et al. (2010). The reproducibility of AFLPs was tested

using 15 replicated samples. Electropherograms were

analysed with Peak Scanner version 1.0 (Applied

Biosystems) with default peak detection parameters.

The minimum ﬂuorescent threshold was set to 50

relative ﬂuorescence units (RFUs). RawGeno version

2.0 (Arrigo et al., 2009), a package for R (R

Development Core Team, 2011), was used for auto-

mated data scoring with the following settings: scoring

range, 75–500 bp; minimum intensity, 50 RFU;

minimum bin width, 1 bp; maximum bin width, 1.5 bp.

Fragments with reproducibility <80% based on

sample replicate comparisons were excluded. The error

rate was calculated as the ratio of mismatches (scoring

of 0 vs. 1) over phenotypic comparisons with AFLP

proﬁles of replicated individuals (Bonin et al., 2004). A

neighbour-joining (NJ) analysis based on Nei–Li

genetic distances (Nei & Li, 1979) was conducted

and bootstrapped (2000 pseudo-replicates) with

TREECON 1.3b (Van de Peer & De Wachter, 1997). A

NeighborNet diagram was produced from a matrix of

uncorrected Pdistances using SplitsTree 4.12 (Huson,

1998). Aprincipal coordinate analysis (PCoA) based on

Jaccard distances was conducted using PAST 2.15

(Hammer, Harper & Ryan, 2001).

PLASTID trnT-trnF SEQUENCING

For 54 individuals (one to ﬁve per population), plastid

DNA was sequenced (Table 1). The 24.5-μL PCR mix

contained 9 μL PCR ReadyMix (Sigma-Aldrich),

0.5 μL (10 μM) each primer (a, c and f; Taberlet et al.,

1991), 1 μL bovine serum albumin (BSA) (1 mg mL−1;

Promega) and 1 μL diluted DNA extract of unknown

concentration. PCR conditions were 5 min at 95 °C,

followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C

and 4 min at 65 °C, followed by a ﬁnal 10-min exten-

sion period at 65 °C. The PCR products were cleaned

with ExoFastAP mixture (Thermo Scientiﬁc) follow-

ing the manufacturer’s instructions. A BigDye Termi-

nator Cycle Sequencing Kit (Applied Biosystems) was

used for cycle sequencing following the manufactur-

er’s instructions, after which electrophoresis was

carried out with an ABI 3100 capillary sequencer

(Applied Biosystems). Sequences were edited with

Geneious 5.5.6 (Biomatters) and aligned manually

using BioEdit 7.0.4.1 (Hall, 1999). The alignment of

the concatenated plastid markers was analysed using

statistical parsimony as implemented in TCS 1.21

(Clement, Posada & Crandall, 2000) with the connec-

tion limit set to 95%; gaps were treated as ﬁfth

character state. For this analysis, indels >1 bp were

reduced to single base pair columns, allowing these

structural mutations to be counted as single base pair

mutations.

ANALYSIS OF NUCLEAR rbcS

An intron region within the nuclear small rubisco

gene (rbcS) was ampliﬁed to test the hybrid origin of

S. bosniaca. As the potential parental taxa S. aria

and S. aucuparia have speciﬁc genotypes for this

nuclear locus, S. bosniaca was expected to show a

combinatory pattern. The rbcS locus was ampliﬁed

using ATGCATGCAGGTGTGGC as forward and GTT-

GTCGAATCCGATGAT as reverse primer (Robertson,

Newton & Ennos, 2004). The PCR mixture (total

volume, 15 μL) contained 0.75 U Taq DNA Polymer-

ase (Qiagen), 1.5 μL 10 × reaction buffer (Qiagen),

0.3 mM GeneAmp dNTP Mix with dTTP (Applied

Biosystems), 0.3 μM each primer and 20–50 ng

genomic DNA. PCRs were performed in an Eppendorf

Mastercycler in the following conditions: denatura-

tion at 95 °C for 3 min, followed by 30 cycles of 1 min

at 94 °C, 1 min at 57 °C and 1 min at 72 °C, and a

ﬁnal extension at 72 °C for 5 min. The PCR products

were separated on 2% agarose gels. Gels were stained

with 0.5 μgmL

−1 ethidium bromide (Sigma-Aldrich),

visualized under UV light and analysed using

Quantum ST4 system (Vilber Lourmat).

ANALYSIS OF NUCLEAR MICROSATELLITES

Ampliﬁcation of four nuclear microsatellite speciﬁc

loci (CH01F02, MSS5, MSS13 and MSS16) was per-

formed for 147 individuals (Table 1), following

Robertson et al. (2010). An ABI PRISM 310 Genetic

Analyzer (Applied Biosystems) was used for electro-

phoretic separation of the PCR products. Alleles were

sized relative to the internal size standard TAMRA

500 (Applied Biosystems). Electropherograms were

analysed using GeneMapper (Applied Biosystems).

Multilocus genetic phenotypes were determined for

each individual in order to analyse genetic diversity

and to determine population structure and origin of

S. bosniaca. The number of multilocus phenotypes

(Ng) was determined and measures of genetic diver-

sity, such as the total number of different alleles seen

across all loci (A′); mean number of different alleles

carried by each individual averaged across all loci

6A. HAJRUDINOVIC

´ET AL.

(H′), proportion of distinguishable genotypes (Ng/N)

(Ellstrand & Roose, 1987) and Simpson’s diversity

index (D), were calculated. For comparison, the same

parameters were calculated for other Sorbus taxa

included in this study.

To estimate allele diversity, the overall number of

different alleles seen across all loci and the mean

number of different alleles carried by each individual

averaged across loci were calculated. Relationships

among multilocus phenotypes were visualized by

PCoA based on Jaccard distances using PAST 2.15

(Hammer et al., 2001).Simulated matings among and

within Sorbus taxa were performed, following

Robertson et al. (2010), to identify the parents of

S. bosniaca.

MORPHOLOGICAL CHARACTERIZATION

Morphological characterization of S. bosniaca was

based on 29 quantitative and eight qualitative charac-

ters scored for 50 adult individuals. The selection of

characters was based on determination keys, formal

descriptions of taxa of subgenus Soraria and our

personal observations (Supporting Information,

Appendix S2). The mean values of ﬁve measurements

per character for each individual were used in descrip-

tive statistics and for the formal description of

S. bosniaca. In order to establish the morphological

distinctness of S. bosniaca, we included the two most

similar tetraploid hybridogenic species, S. hybrida and

S. borbasii, and the diploid hybrid S. × thuringiaca

reported from the Balkans. Character scores of

S. hybrida and S. × thuringiaca were taken from Rich

et al. (2010). A comparative review of character varia-

tion is given in Appendix S2.

RESULTS

FLOW CYTOMETRY

The results of genome size determinations are pre-

sented in Table 2. Accessions from sampling site 6

(Innsbruck, Austria) collected in winter could not be

analysed. Mean values of nuclear DNA content

ranged from 1.30 pg in diploid S. aria to 2.98 pg in

tetraploid S. hybrida. Sorbus aria included di-, tri-

and tetraploid cytotypes, whereas all other taxa were

uniform: S. aucuparia and S. ×thuringiaca were

diploid, and S. austriaca,S. bosniaca and S. hybrida

were tetraploid.

FLOW CYTOMETRIC SEED SCREEN AND

POLLEN VIABILITY

Thirty-four seeds from 13 individuals of S. bosniaca

were analysed to infer the reproduction mode. Twenty-

four seeds showed apomictic cytometric proﬁles with

clear peaks in histograms representing tetraploid

embryo and dodecaploid endosperm (Appendix S1).

Such an embryo/endosperm proﬁle indicates fertiliza-

tion of the endosperm (pseudogamy) with one unre-

duced tetraploid or two reduced diploid spermal cell(s),

whereas the embryo developed parthenogenetically.

Four seeds had clear tetraploid embryo peaks, but the

endosperm was 11x,13xor 15x, making the interpre-

tation of its origin difficult (Appendix S1). Two seeds

showed a series of peaks corresponding to endoredu-

plication of a tetraploid embryo, but no endosperm was

evident, and in four seeds no peaks were visible. The

percentage of viable pollen ranged from 62% to 97%

(mean, 87% ± 12%; median, 94%).

AFLP

We obtained 362 high-quality and reproducible

AFLP fragments from 144 individuals. The initial

average error rate was 5.6%. The NJ analysis

revealed eight major clusters with high bootstrap

support (BS) between 93% and 100%; the same

groups were visualized by the NeighborNet diagram.

The NeighborNet diagram complemented with BS

values is shown in Figure 1A. The highly divergent

clusters corresponded to S. aucuparia,S. × thuringi-

aca, diploid S. aria, triploid and tetraploid S. aria,

two tetraploid S. austriaca groups with BS values of

99% and 100%, S. bosniaca and S. hybrida. PCoA

discriminated the same entities (Fig. 2A). The ﬁrst

two axes of the PCoA plot, cumulatively explaining

50% of the total variance, discriminated between

S. aucuparia and S. aria and positioned hybrid and

polyploid apomictic accessions in separate groups

(Fig. 2A).

PLASTID trnT-trnF SEQUENCING

The trnT-trnF sequences (concatenated trnT-trnL

intergenic spacer, GenBank accessions KP794806–

KP794831, and trnL-trnF partial sequence, GenBank

accessions KP794832–KP794857) were 1742 bp

(S. aucuparia from sampling site 6) to 1863 bp (S. aria

from sampling site 1) long, resulting in an alignment of

1933 bp with 30 variable characters (16 indels and 14

nucleotide substitutions). No intrapopulational vari-

ability was detected. Eleven substitutions and nine

indels contributed to the separation into two groups,

one corresponding to S. aria and the other to S. aucu-

paria and all other taxa. In the TCS analysis, the two

groups failed to connect at a connection limit of 95%.

The S. aucuparia haplotype group comprised four hap-

lotypes: two S. aucuparia individuals were separated

from the central haplotype by one (sampling site 6) or

two (sampling site 1) indels, and S. bosniaca differed in

one nucleotide substitution and one insertion (Fig. 1B).

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 7

-0.1

-0.2

-0.3

0.1

0.2

0.3

0.4

-0.1 -0.2 -0.3 -0.40.40.5 0.3 0.2 0.1

PCo 1 (20.4%)

PCo 2 (12.3%)

0.06

0.12

0.18

0.24

-0.06

-0.12

-0.18

-0.24

0.1 0.2 0.3 0.4-0.4-0.5 -0.3 -0.2 -0.1

PCo 1 (37.3%)

PCo 2 (12.7%)

Figure 2. Relationships among European Sorbus illustrated by a principal coordinate analysis of ampliﬁed fragment

length polymorphism (AFLP) data (A) and nuclear microsatellite data (B) coded as multilocus phenotypes. Symbols are

as in Figure 1, except for additional samples of S. borbasii marked with a cross in (B).

8A. HAJRUDINOVIC

´ET AL.

The S. aria haplotype group comprised six haplotypes;

most samples exhibited a central haplotype from

which ﬁve rare haplotypes were separated by one to

three nucleotide substitutions and/or indels (Fig. 1B).

ANALYSIS OF NUCLEAR rbcS

The combination of parental bands observed in

S. bosniaca was identical to that of other analysed

hybrid taxa and supported their hybrid origin from

S. aria and S. aucuparia (Supporting Information,

Appendix S3).

ANALYSIS OF NUCLEAR MICROSATELLITES

The total number of different alleles scored across all

loci (A′) for S. bosniaca was the lowest compared with

the other analysed polyploid taxa from Bosnia and

Herzegovina (sampling sites 1–4). In contrast, the

mean number of different alleles carried by each

individual averaged across all loci (H′) was relatively

high for this taxon (Table 3). The maximum number

of alleles per locus in S. bosniaca was four (CH01F02,

Supporting Information, Appendix S4). At the same

locus, alleles 186 and 188 were found to be speciﬁc for

S. aucuparia, and alleles 196 and 200 originated from

S. aria (Supporting Information, Appendix S5).

Each diploid individual of S. aucuparia and S. aria

had a unique multilocus phenotype (Table 3). Most

analysed polyploid taxa had at least a few individuals

with the same multilocus phenotype, indicating a

clonal origin (Appendix S4). Low clonal diversity was

detected in S. bosniaca (Table 3); only two individuals

had a multilocus genotype differing at the locus MSS5

(Appendix S4) from the genotype retrieved from the

remaining 18 individuals. PCoA showed strong diver-

gence of diploid sexual S. aria and S. aucuparia,

whereas all hybrid and polyploid taxa had distinct

intermediate positions (Fig. 2B). Sorbus bosniaca was

clearly separated from all other analysed taxa.

Mating simulations identiﬁed diploid S. aria and

S. aucuparia as the most probable parental taxa of

S. bosniaca (Supporting Information, Appendix S6).

MORPHOLOGY

A comparative review of morphological character vari-

ation of species of subgenus Soraria with similar

morphology showed speciﬁc discriminating features of

S. bosniaca (Appendix S2). Sorbus bosniaca and

S. hybrida markedly differed in maximum number of

lateral leaf veins (15–18 in S. bosniaca vs.14–23 in

S. hybrida, 5%–95% ranges), shape of the lamina apex

(acute vs.truncate or rounded to obtuse), fruit shape

(globose to longer than wide vs.globose to wider than

long) and the point of fusion of styles (divided to the

base vs.fused nearly to the top). Sorbus bosniaca and

S. borbasii differed in lamina length (7–10 cm in

S. bosniaca vs.6–8 cm in S. borbasii), lamina shape

(ovate to elliptic vs.broadly ovate), number of lateral

leaf veins (15–18 vs.15–24) and serration of the

leaﬂets (uniserrate with acuminate teeth pointing

towards the apex vs.entire with few teeth at the leaﬂet

apex). Differences with S. × thuringiaca were found in

lamina length (7–10 cm in S. bosniaca vs.7–13 cm in

S. × thuringiaca), lamina width (4–6 cm vs.4–8 cm),

lamina shape (ovate to elliptic vs.oblong to oblong-

lanceolate), number of lateral leaf veins (15–18 vs.

16–27), fruit length (0.8–1.0 cm vs.1.0–1.4 cm), fruit

Table 3. Nuclear microsatellite-derived genetic diversity measures for the analysed Sorbus taxa or cytotypes

Taxon DNA ploidy NX

eXoA′H′NgNg/ND

Diploids

S. aria 2x31 2 2 35 1.78 31 1.00 –

S. aucuparia 2x35 2 2 32 1.44 35 1.00 –

S. × thuringiaca 2x1 2 2 8 2.00 – – –

Polyploids

S. aria 3x16 3 3 21 2.34 8 0.50 0.86

S. aria 4x12 4 4 24 2.75 10 0.83 0.95

S. austriaca 4x27 4 4 19 2.99 2 0.07 0.40

S. borbasii – 3 – 4 12 2.50 1 0.33 0.00

S. bosniaca 4x20 4 4 13 2.75 2 0.10 0.19

S. hybrida 4x2 4 3 8 2.00 1 0.50 0.00

N, number of individuals sampled; Xe, expected ploidy, based on genome size data; Xo, observed ploidy, based on the

maximum number of displayed alleles at a single locus; A′, total number of different alleles seen across all loci; H′, mean

number of different alleles carried by each individual averaged across all loci; Ng, number of multilocus phenotypes

detected; Ng/N, proportion of clones detected; D, estimate of multilocus phenotype diversity (multiclonal species only).

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 9

width (0.8–0.9 cm vs.0.8–1.4 cm) and the colour of

anthers (pale pink vs.cream to yellow).

DISCUSSION

The joint application of ﬂow cytometry and a suite of

molecular methods revealed a clear structure among

accessions of Sorbus subgenus Soraria and its paren-

tal subgenera Aria and Sorbus from Bosnia and Her-

zegovina and elsewhere. Two genetic groups are

evident (Fig. 1A) within both S. aria and S. austriaca

(Fig. 2B, Appendix S4). In the former taxon, tri- and

tetraploid cytotypes yielded eight and ten distinct

microsatellite-derived multilocus phenotypes, respec-

tively (Fig. 2B, Appendix S4), but the AFLP separa-

tion did not receive bootstrap support (Figs 1A, 2A).

In contrast, there was clear AFLP divergence of indi-

viduals within the S. austriaca group (Fig. 1A). Sub-

divisions within the polyploid taxa might reﬂect

independent hybridization events followed by repro-

ductive isolation via apomixis (Robertson et al., 2010).

Ongoing studies (A. Hajrudinovic´, unpubl. data) will

explore the cryptic diversity within S. austriaca and

S. aria.

The genetic data and a comparative review of mor-

phological characters consistently conﬁrmed the dis-

tinct position of S. bosniaca within subgenus Soraria,

which encompasses both hybrid sexual and apomictic

taxa disjunctly distributed across Europe and Asia

(Májovský & Bernátová, 2001). The hybrid origin

of S. bosniaca is apparent both from morphology

(Figs 3, 4) and molecular data (AFLPs, microsatel-

lites, rbcS genotype: Figs 1A, 2; Appendices S3, S4).

Genome size data revealed consistent tetraploidy

(Table 2), and mating simulations based on micros-

atellite data suggested that S. bosniaca resulted from

hybridization between diploid S. aria and S. aucu-

paria (Appendix S6). However, our simulation

approach requires a complete sampling of parents,

which could not be achieved, and therefore we cannot

rule out the alternative scenario of hybridization of

an apomictic polyploid and a sexual diploid progeni-

tor (Robertson et al., 2010). Plastid DNA sequence

data revealed that the single haplotype found in

S. bosniaca is closely related to those detected in

S. aucuparia, suggesting this species as female

parent, as is characteristic for most taxa of subgenus

Soraria (Chester et al., 2007; Robertson et al., 2010).

However, S. bosniaca possesses a private haplotype

derived from the most common S. aucuparia haplo-

type by one indel and one substitution (Fig. 1B), sug-

gesting mutations after the origin of S. bosniaca;

alternatively, the haplotype is present in S. aucu-

paria, but was not sampled. The mating simulation

test identiﬁed diploid S. aria as the male parent of

S. bosniaca, which is in line with S. aria being the

most common pollen donor in hybrid events in the

genus (Nelson-Jones, Briggs & Smith, 2002; Chester

et al., 2007).

Most polyploid Sorbus are pseudogamous apomicts

(Liljefors, 1953; Rich et al., 2010; Lepší et al., 2013;

Ludwig et al., 2013), which is also the case in S. bos-

niaca, as supported by ﬂow cytometric seed screen

(FCSS) proﬁles (Appendix S1). As tetraploid Sorbus

taxa are self-compatible (Ludwig et al., 2013) and

S. bosniaca produces viable pollen, self-fertilization of

the endosperm probably facilitates long-term persis-

tence of isolated populations. Apomixis in S. bosniaca

is also supported by the low genetic diversity meas-

ures derived from nuclear microsatellites (Table 3).

According to Robertson et al. (2010), low values of A′

for polyploid taxa suggest a monophyletic origin fol-

lowed by apomixis, whereas high values of H′are in

accordance with polyploids being the progeny of

genetically divergent parents. Visual inspection of

AFLP electropherograms of S. bosniaca with the soft-

ware GENOGRAPHER 1.6.0 (J. J. Benham, version

no longer available) indicated that the slight diver-

gence among AFLP phenotypes (Figs 1A, 2A) is arte-

factual and results from errors in the automated

scoring procedure caused by varying peak heights

(Supporting Information, Appendix S7). Microsatel-

lites identiﬁed two multilocus phenotypes in S. bos-

niaca (Fig. 2B), based on one locus (Appendix S4).

The inspection of morphological variation between

the individuals from the two groups did not show any

signiﬁcant differences (data not shown). The occur-

rence of different clonal genotypes may be a result of

either independent hybridization events in the past or

mutations in clonal genotypes.

Several northern European apomicts are morpho-

logically similar to S. bosniaca, but differ in their

parentage (S. ×liljeforsii T.C.G.Rich; Rich, 2007)

or have narrow distributions (S. pseudofennica

E.F.Warb; Robertson et al., 2004). Sorbus hybrida is

a common tetraploid representative of subgenus

Soraria in Scandinavia, which has similar parentage

to S. bosniaca. It originated from hybridization

between S. aucuparia and tetraploid S. rupicola

(Syme) Hedl., a widespread member of the S. aria

aggregate in northern Europe (Rich et al., 2010). Two

geographically closer species with relatively similar

morphology and origin are S. ×thuringiaca and

S. borbasii. The former has been reported from Mac-

edonia (as S. ×semipinnata; Micevski, 1998), albeit

without ploidy information. Elsewhere, S. × thuringi-

aca is considered as a diploid ﬁrst-generation hybrid

between S. aria s.s. and S. aucuparia (Robertson

et al., 2010). Sorbus borbasii, however, is endemic to

Bulgaria and Romania (Ciocârlan, 1988; Delipavlov

et al., 2003), and it has been proposed that it origi-

nated through hybridization between S. graeca

10 A. HAJRUDINOVIC

´ET AL.

(Spach) Kotschy (synonym S. cretica Lindl.; belonging

to the S. aria aggregate sensu Pellicer et al., 2012)

and S. aucuparia (Jávorka, 1915). Our microsatellite

data suggest that S. borbasii from the locus classicus

(Mt. Domugled, Romania) is tetraploid, as is S. bos-

niaca, but differs in three of the four analysed micro-

satellite loci (Appendix S4). Genetic uniformity of the

three analysed individuals of S. borbasii suggests an

apomictic mode of reproduction.

Altogether, several lines of evidence strongly

suggest that S. bosniaca is a new, narrowly endemic

species resulting from an independent hybridization

event accompanied by tetraploidization and a switch

to apomixis.

BCD

1 cm

Figure 3. Illustration of Sorbus bosniaca. A, Flowering long shoot bearing a lateral sterile short shoot. B, Flower in

longitudinal section. C, Flower from above. D, Ripe pome.

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 11

Figure 4. Holotype of Sorbus bosniaca.

12 A. HAJRUDINOVIC

´ET AL.

SORBUS BOSNIACA A.HAJRUDINOVIC

´,FRAJMAN,

SCHÖNSW.&F.BOGUNIC

´SP.NOV.

Diagnosis

Sorbus bosniaca is a hybridogenous tetraploid apom-

ictic species, which differs from the morphologically

similar S. borbasii Jav. in lamina shape (ovate to

elliptic vs. broadly ovate), lamina length (7–10 cm vs.

6–8 cm), number of lateral leaf veins (15–18 vs.

15–24) and serration of the leaﬂets (uniserrate with

acuminate teeth pointing towards the apex vs. almost

entire with few teeth at the leaﬂet apex). It differs

from S. hybrida in number of lateral leaf veins (15–18

vs. 14–23), shape of the lamina apex (acute vs. trun-

cate or rounded to obtuse), fruit shape (globose to

longer than wide vs. globose to wider than long) and

the point of fusion of styles (divided to the base vs.

fused nearly to the top). Sorbus bosniaca differs from

S. × thuringiaca in lamina shape (ovate to elliptic vs.

oblong to oblong-lanceolate), lamina length (7–10 cm

vs. 7–13 cm), lamina width (4–6 cm vs. 4–8 cm),

number of lateral leaf veins (15–18 vs. 16-27), fruit

length (0.8–1 cm vs. 1–1.4 cm), fruit width (0.8–0.9

cm vs. 0.8–1.4 cm) and the colour of anthers (pale

pink vs. cream to yellow).

Holotype

Bosnia and Herzegovina, slopes and plateau of

Mt. Krug planina, 4 km east of village Šujica

(western Bosnia and Herzegovina), 1300 m altitude,

43°50′32″N, 17°11′59″E, 9.10.2012, A. Hajrudinovic´&

F. Bogunic´. Holotype: WU (080424); Isotype: SARA

(51405).

Description

Shrub or small polycormous tree up to 5 m, with

rounded crown. Trunks up to 30 cm in diameter. Buds

lanceolate, dark reddish-brown, white villous. Bark

grey, smooth. Broad leaves of short sterile shoots

(6.1–)6.9–9.9(–10.2) cm, (1.3–)1.5–1.8(–1.9) times as

long as wide, ovate to elliptic, rarely obovate, widest

at (39–)42–53(–62)% of the lamina, with one to four

free, narrowly elliptic, acute basal leaﬂets in opposite

or sub-opposite pairs and an ovate, lobed terminal

leaﬂet which becomes less lobed towards the acute

apex. Leaf margin more or less uniserrate with small,

acuminate teeth pointing towards the apex, but entire

near the midrib. Upper leaf surface coriaceous, green,

lower surface greyish-green to greyish-white tomen-

tose, with (14–)15–18(–19) veins held at an angle of

(33–)42–56(–57)° to the midrib. Petioles (1.3–)1.5–

2.3(–2.4) cm, greenish or reddish, white villous. Inﬂo-

rescence a moderately lax corymb, (4.7–)4.8–6.9 cm in

diameter, with white villous branchlets and pedicels.

Sepals triangular, green, white villous adaxially, with

or without brownish glands on the margins. Petals

white, 0.71–0.82(–0.83) cm long, 0.53–0.61 cm wide,

ovate, white villous adaxially near the base. Anthers

pale pink. Styles two or three (four), divided to the

base. Ripe fruits (0.81–)0.83–0.96(–0.97) cm long,

(0.73–)0.77–0.91(–0.96) cm wide, globose to ellipsoid,

scarlet, with a few small scattered lenticels. Seeds

3–4 mm, ellipsoid, brown, one to two per fruit.

Genome size 2.69 pg, corresponding to the tetraploid

level. Reproduction apomictic. Pollen viability c. 87%.

Phenology

Sorbus bosniaca ﬂowers in June and the fruits ripen

from September to October.

Etymology

The epithet bosniaca refers to Bosnia (as a geographi-

cal part of Bosnia and Herzegovina), where the new

species has its locus classicus.

Distribution and ecology

The species inhabits the slopes and plateau of Mt.

Krug planina, which is situated c. 4 km east of the

village of Šujica, north-west of Livno (western Bosnia

and Herzegovina). The species covers an area of

c.1000 ha with an altitudinal range from 1240 to

1380 m. The total number of adult individuals is c. 300.

Plants thrive in a mosaic of mixed stands of Fagus

sylvatica L. and Abies alba Mill. in various stages of

degradation (coppices and scrublands) and different

associations of xerophilous meadows on calcareous-

dolomitic substrate. The degraded sites mostly have a

south-east exposure, which is dry and sunny, and

characterized by mosaics of shallow stony soils (molic

leptosols and calcic cambisols).

Conservation status

According to our best knowledge, the occurrence of

S. bosniaca is restricted to Mt. Krug planina and

Koric´ino sedlo (c. 25 km distance from Mt. Krug

planina, unveriﬁed because of land mines). Thus,

following the criteria of IUCN (2014), this species

should be designated as Vulnerable, matching the

criteria D1 (the population size is estimated to be

<1000 mature individuals) and D2 (the area of occu-

pancy is very restricted, typically <20 km2, or exists

at typically ﬁve or fewer locations, and there is a

plausible natural or anthropogenic threat).

Conventional species-based conservation efforts

may fail to preserve the diversity in taxonomically

complex groups such as Sorbus, the dynamic evolution

of which requires different approaches (Ennos, French

& Hollingsworth, 2005). A new concept, termed the

Processes-Based Species Action Plan for taxonomically

complex groups, was introduced by Ennos et al. (2012)

using Sorbus species in the Glens of Arran (Scotland,

UK) as the study model. This concept was designed

POLYPLOID SORBUS FROM BOSNIA AND HERZEGOVINA 13

with the purpose of conserving the processes generat-

ing biodiversity and sustaining or increasing the diver-

sity and numbers of interacting lineages. The type

locality of S. bosniaca, Mt. Krug planina, hosts several

potentially interacting sexual diploid and polyploid

apomictic Sorbus lineages (Table 1), which might gen-

erate new biodiversity through hybridization and

backcrossing. Therefore, the area should be designated

as an area of particular conservation concern. Alto-

gether, as supported by the present study and forth-

coming analyses (A. Hajrudinovic´, F. Bogunic´, P.

Schönswetter, B. Frajman, S. Siljak-Yakovlev, unpubl.

data), the Balkans should be regarded as the fourth

European hotspot of diversiﬁcation and speciation in

Sorbus.

ACKNOWLEDGEMENTS

This work was supported by a One-Month-Visit

research scholarship to A. H. granted by the Austrian

Agency for International Cooperation in Education &

Research and ﬁnanced by the Austrian Federal Min-

istry of Science and Research, and by a visit grant

to F. B. by the University of Innsbruck. Laboratory

expenses of AFLP and plastid sequencing analyses

were covered by the University of Innsbruck. In addi-

tion, the study was supported by grants of the

Environmental Fund of the Federation Bosnia and

Herzegovina (grant no. 01-02-1053/2012) and the Min-

istry of Education and Science of Federation of Bosnia

and Herzegovina (grant no. 05-14-4565-1/12) to F. B.

We thank Spencer Brown and Mickaël Bourge for their

assistance on the IBiSA Cytometry Platform of the

Imagif Cell Biology Unit of the Gif campus (http://

www.imagif.cnrs.fr). Mike Fay supported us with

samples from the UK, Finland and Austria; Csaba

Németh with samples from Romania; and Zoltán

Barina and Antun Alegro provided photographs of

S. borbasii from BP. Adnan Medic´ helped in the ﬁeld

collecting samples from Bosnia and Herzegovina. We

thank Daniela Pirkebner and Marianne Magauer,

who supported the molecular genetic laboratory

work.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s website:

Appendix S1. Flow cytometric seed screen (FCSS) of Sorbus bosniaca.

Appendix S2. Comparative review of morphological character variation of similar Soraria taxa.

Appendix S3. Electrophoretic rbcS banding patterns for Sorbus aucuparia,S. austriaca,S. bosniaca,

S. hybrida,S. × thuringiaca and S. aria s.s. M, 100-bp size marker.

Appendix S4. Multilocus phenotypes for analysed polyploid Sorbus taxa.

Appendix S5. Allele compositions of the analysed sexual Sorbus taxa in the four analysed microsatellite loci.

Appendix S6. Simulated matings for Sorbus bosniaca.

Appendix S7. Aligned ampliﬁed fragment length polymorphism (AFLP) electropherograms of Sorbus bosniaca

(three selective primer combinations) visualized with the software GENOGRAPHER 1.6.0.

16 A. HAJRUDINOVIC

´ET AL.

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Feb 2023

The interplay of polyploidisation, hybridization, and apomixis contributed to the exceptional diversity of Sorbus (Rosaceae), giving rise to a mosaic of genetic and morphological entities. The Sorbus austriaca species complex from the mountains of Central and South-eastern Europe represents an allopolyploid apomictic system of populations that originated following hybridisation between S. aria and S. aucuparia. However, the mode and frequency of such allopolyploidisations and the relationships among different, morphologically more or less similar populations that have often been described as different taxa remain largely unexplored. We used amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequencing, and analyses of nuclear microsatellites, along with multivariate morphometrics and ploidy data, to disentangle the relationships among populations within this intricate complex. Our results revealed a mosaic of genetic lineages—many of which have not been taxonomically recognised—that originated via multiple allopolyploidisations. The clonal structure within and among populations was then maintained via apomixis. Our results thus support previous findings that hybridisation, polyploidization, and apomixis are the main drivers of Sorbus diversification in Europe.

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Jul 2020

Hybridization associated with polyploidization and apomixis is a frequent mechanism of speciation. Sorbus is a genus with ongoing hybridization resulting in a polyploid complex with different parental lineage. Triparens is the smallest hybridogenous subgenus of Sorbus so far known to comprise only two taxa, S. intermedia and S. × liljeforsii that combine the genomes of three taxa (S. aria agg., S. aucuparia and S. torminalis). To elucidate the origins of S. dacica, S. paxiana and S. tauricola, three new trigenomic candidates formerly believed to be of biparental origin with either S. aria agg. × S. aucuparia or S. aria agg. × S. torminalis lineage we combined data from HPLC and chloroplast DNA analysing additional 33 related taxa as well. We concluded that the ‘torminalis-type’ flavonoid profile and the ‘aucuparia-type’ plastid indicate the participation of both S. torminalis and S. aucuparia resulting in the formation of S. dacica, S. paxiana and S. tauricola. Sorbus aria agg. as the third ancestor and as a necessary link to meet genes of S. torminalis and S. aucuparia in one genome is obvious from morphological features (densely tomentose undersides of leaves). The tetraploid cytotypes and obligate pseudogamy of S. dacica and S. paxiana were determined by flow cytometry and are published here for the first time. The most probable evolutionary scenario for Triparens species is: 1. a diploid sexual S. aucuparia as pollen acceptor hybridized with a tetraploid apomictic taxon from the S. aria agg. producing a triploid apomictic taxon with ‘aucuparia-type’ plastid inherited maternally; 2. during a second crossing event this subgenus Soraria hybrid as maternal progenitor hybridized with the sexual diploid S. torminalis (providing gene(s) of apigenin O-glucuronide synthesis) forming a tetraploid Triparens hybrid with ‘aucuparia-type’ plastid and ‘torminalis-type’ flavonoids.

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May 2021

Various taxonomic concepts of the classification of Cotoneaster species from the area of the Western Carpathians were proposed throughout the years, based on several methodological approaches. In this paper we try to find out, whether it is possible to distinguish these species based on the absolute genome size data. The absolute genome size of 143 Cotoneaster individuals (32 C. integerrimus, 94 C. melanocarpus agg. and 17 C. tomentosus) was estimated by flow cytometry. Samples were collected from 45 localities within the area of the Western Carpathians. Flow cytometry revealed a significant difference in genome size between pentaploid C. tomentosus (3.15 ± 0.06 pg) and both tetraploid species C. integerrimus (2.37 ± 0.08 pg) and C. melanocarpus agg. (2.36 ± 0.06 pg). Statistically not significant difference in genome size was recorded between C. integerrimus and C. melanocarpus agg.

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Apr 2023
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Apr 2022

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May 2021
COMP CYTOGENET

Eight taxa of Sorbus Linnaeus, 1753 sensu stricto (Rosaceae) from China have been studied karyologically through chromosome counting, chromosomal measurement and karyotype symmetry. Genome size was also estimated by flow cytometry. Six taxa, S. amabilis Cheng ex T.T.Yu et K.C.Kuan, 1963, S. hupehensis var. paucijuga (D.K. Zang et P.C. Huang, 1992) L.T. Lu, 2000, S. koehneana C.K. Schneider, 1906, S. pohuashanensis (Hance, 1875) Hedlund, 1901, S. scalaris Koehne, 1913 and S. wilsoniana C.K. Schneider, 1906 are diploids with 2n = 34, whereas two taxa, S. filipes Handel-Mazzetti,1933 and S. ovalis McAllister, 2005 are tetraploid with 2n = 68. In general, the chromosome size is mainly small, and karyotypes are symmetrical with predominance of metacentric chromosomes. Genome size variation of diploids and tetraploids is 1.401 pg –1.676 pg and 2.674 pg –2.684 pg, respectively. Chromosome numbers of S. amabilis and S. hupehensis var. paucijuga, and karyotype and genome size of eight taxa studied are reported for the first time. This study emphasised the reliability of flow cytometry in genome size determination to infer ploidy levels in Chinese native Sorbus species.

Genome size variation and karyotype diversity in eight taxa of Sorbus sensu stricto (Rosaceae) from China

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May 2021

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Dec 2023

In overlapping distribution areas of Sorbus pohuashanensis and S. discolor in North China (Mount Tuoliang, Mount Xiling and Mount Baihua), Sorbus individuals were found with pink fruit, which have never been recorded for the flora of China. Fourteen morphological characters combined with four chloroplast DNA markers and internal transcribed spacer (ITS) were used to analyze the origin of the Sorbus individuals with pink fruits and their relationship to S. pohuashanensis and S. discolor . PCA, SDA and one-way (taxon) ANOVA of morphological characters provided convincing evidence of the hybrid origin of Sorbus individuals with pink fruits based on a novel morphological character and many intermediate characters. Haplotype analysis based on four cpDNA markers showed that either S. pohuashanensis or S. discolor were maternal parents of Sorbus individuals with pink fruits. Incongruence of the position of Sorbus individuals with pink fruits between cpDNA and ITS in cluster trees supported by DNA sequence comparative analysis, implying former hybridization events between S. pohuashanensis and S. discolor . Multiple hybridization events between S. pohuashanensis and S. discolor might have contributed to the generation of Sorbus individuals with pink fruits. This study has provided insights into hybridization between species of the same genus in sympatric areas, which is of great significance for the study of interspecific hybridization.

OBRAZOVANJE, ISTRAŽIVANJE I ODRŽIVO UPRAVLJANJE ŠUMSKIM RESURSIMA KAO FAKTOR ODRŽIVOG RAZVOJA U BOSNI I HERCEGOVINI / EDUCATION, RESEARCH AND SUSTAINABLE FOREST MANAGEMENT AS A FACTOR OF SUSTAINABLE DEVELOPMENT IN BOSNIA AND HERZEGOVINA

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Jul 2023

The sustainable forest management concept contains the essence of the sustainable development paradigm - meeting the current needs of society without compromising the needs of future generations. The strategic commitment of the Faculty of Forestry University of Sarajevo is aimed at quality education of forestry experts, conducting innovative research and the most intensive cooperation with the economy. The methodological approach in this paper is based on a critical analysis of the scope of the educational-research process at the Faculty of Forestry University of Sarajevo, and the potentials of sustainable forest management in BiH, to determine to what extent higher education and research on environmental, sociological and economic aspects of forest management can affect positive changes in BiH society. Apart from identifying the benefits of forest ecosystems in the context of contributing to sustainable development, the research on the attitudes of teaching staff related to the contribution of the educational-research process to general goals of sustainable development from the 2030 Agenda was conducted. The results of this paper show that the concept of sustainable forest management, as it is realized in educational-research process at the Faculty of Forestry University of Sarajevo, has significant potential to contribute to sustainable development in BiH. This contribution is reflected in biodiversity conservation, mitigation of climate change, carbon storage, prevention of natural disasters, impact on the stability of water and soil, energy supply from renewable sources, continuous economic growth, socially responsible production and consumption, job security, development of rural and urban areas as well as maintaining and improving the psychophysical health of the population. Based on the obtained results, it can be concluded that sustainable forest management, grounded on scientific postulates, understanding of diversity of forest ecosystem services and dynamics in society's requirements towards forests, multidisciplinary and cross-sectoral cooperation, can be an important factor of sustainable development in BiH.

Reproduction Modes and Conservation Implications in Three Polyploid Sorbus Stenoendemics in Eastern Slovakia (Central Europe)

Article

Full-text available

Jan 2023

The remarkable species diversity of the genus Sorbus is a result of polyploidization and frequent hybridization between interacting species of different cytotypes. Moreover, hybridization is possible between several parental taxa. Gametophytic apomixis, which is common among polyploid Sorbus taxa, indicates the role of clonal reproduction in the evolutionary stabilization of hybridogeneous genotypes. The precise determination of the origin of seeds and their quantitative evaluation may elucidate inter-cytotype interactions, the potential role of mixed-cytotype populations in evolutionary success, and the long-term survival of some hybrid species. We investigated the reproduction modes of selected species of Sorbus in mixed-cytotype populations in eastern Slovakia, Central Europe. We determined the pollen quality, seed production rate, and the ploidy level of mature trees, as well as the origin of the embryo and endosperm in seeds of the stenoendemics S. amici-petri, S. dolomiticola, and S. hornadensis. The tetraploids S. amici-petri and S. hornadensis are characterized by regular and highly stainable pollen grains and reproduce predominantly via pseudogamous apomixis. In contrast, triploid S. dolomiticola usually has oval, heterogenous, and weakly stainable pollen grains, suggesting male meiotic irregularities. Although seeds originate via pseudogamous apomixis in S. dolomiticola as well, the ploidy level of sperm cells participating in the fertilization of central cells is usually determined by co-occurring species of different cytotypes. This suggests that maintaining mating partners is necessary for the long-term survival of a triploid species. We documented rare BIII hybrids and the residual sexuality in tetraploids. The distribution of seeds of meiotic and apomeiotic origins in S. amici-petri shows bimodal characteristics; however, genotypes with predominantly sexual seed types are rare. Reproduction modes documented in polyploid stenoendemics of Sorbus and inferred microevolutionary intercytotype relationships highlight the mixed-cytotype populations as the source of biodiversity in apomictic plant complexes. We suggest that conservation efforts should focus on maintaining the species and cytotypic diversity of Sorbus populations, especially when it comes to the conservation of triploid species.

Pattern and Process in Balkan Biodiversity — An Overview

Chapter

Full-text available

Jan 2004

The morose manner in which the grand vizier, Jusuf Ibrahim, is reported by the celebrated writer, the Bosnian Serb, No Andrić, to have recalled his Bosnian homeland probably reflects equally the opinions of the two men. In the 19th century, Ibrahim had been transferred from Bosnia in his early childhood to Istanbul, the most splendid city of its time, as part of a `harač’, or tax in blood, which was paid in the form of children by Christian families for them to be converted to Islam and trained as yanitsarees. Jusuf Ibrahim, the poor Bosnian child, went on to attain the highest position in the Ottoman’s court. Both men made successful careers in the outside world, but both apparently harboured deep-seated resentment about their early childhoods in Bosnia.

Sorbus latifolia (Rosaceae): nova vrsta u flori Bosne i Hercegovine / Sorbus latifolia (Rosaceae): A New Species in the Flora of Bosnia and Herzegovina

Conference Paper

Full-text available

Jan 2012

Sorbus×liljeforsii, a name for the S. aucuparia×intermedia hybrid (Rosaceae)

Article

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Dec 2007
NORD J BOT

Tim Rich

The uncommon spontaneous hybrid between Sorbus aucuparia L. and S. intermedia (Ehrh.) Pers (Rosaceae) is described and named as Sorbus×liljeforsii.

Contributions to the Knowledge of the Flora of the Dinarides (Balkan Peninsula)

Article

Full-text available

May 2014
PHYTON-ANN REI BOT A

The Balkan Peninsula belongs to the botanically least explored regions of Europe, even if it is probably Europe's floristically richest area. During several excursions to the western Balkan Peninsula we encountered new and noteworthy localities of rare and endangered plant species and hence present our most significant findings. We discovered a second locality of the recently described Androsace komovensis SCHÖNSW. & SCHNEEW. (Primulaceae) on the summit of Mt. Kërrshat e Mdh'aj (Alpet Shqiptare/Prokletije/Albanian Alps) at the border between Montenegro and Kosovo. Similarly to the type locality, the newly discovered population is spatially very restricted and individual-poor. The occurrence of Doronicum grandiflorum LAM. (Asteraceae-Senecioneae) on the Balkan Peninsula has not been clear so far. Whereas the species' suggested presence in Albania or Montenegro appears to be based on misidentifications, we confirm its existence in the Čvrsnica and Prenj mountain ranges in Bosnia and Herzegovina and provide characters to distinguish it from D. columnae TEN. and D. orientale HOFFM., for which it has been commonly mistaken. We provide an updated distribution map of Heliosperma macranthum PANČIĆ (Caryophyllaceae-Sileneae), an endemic of the Alpet Shqiptare/Prokletije/Albanian Alps, based on our own records, herbarium and literature data. Poa glauca VAHL subsp. frearitis (ORPH. ex HALÁCSY) H. SCHOLZ (Poaceae-Poeae) is a little-known taxon of the arctic-alpine Poa glauca complex, previously only known from Mt. Varnous and Mt. Smolikas in Greece. We here report the first record for Kosovo from the summit of Mt. Gjeravica/Deravica (Alpet Shqiptare/Prokletije/Albanian Alps), situated at least 200 km NNW of the previously known occurrences in southeastern Europe.

Evolutionary significance of apomixis in the genus Sorbus (Rosaceae

Article

Jan 1993

A. Jankun

Apomicts in the genus Sorbus have been reported almost throughout its range of distribution, in Asia, Europe and North America. However, embryological proof of apomixis has been established only for Scandinavian, Central European and Armenian taxa. Apomixis continues to play an important role in the evolution of Sorbus by stabilising both autopolyploid primary taxa and those of hybrid origin. -from English summary

R: A Language and Environment for Statistical Computing

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Jan 2011

R Development Core Team

Flora ilustrată a României

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Jan 2000

V. Ciocârlan

Guidelines for Using the IUCN Red List Categories and Criteria. Version 8.1

Book

Jan 2010

IUCN Standards and Petitions Subcommittee

Sorbus madoniensis (Rosaceae), a new species from Sicily

Article

Jan 2012

Sorbus madoniensis, a new species of Sorbus sect. Aria, is described, so far known only from two neighbouring localities on Mt. Carbonara (Madonie mountains, Sicily). It is related to S. graeca, which also occurs in the Madonie, and indeed shares with S. madoniensis one of its localities.

The role of hybridization in plant speciation

Article

Jan 2009

Pam Soltis

... [Because an autopolyploid may arise via crossing between genetically different individuals (179, 195), the concept of hybridization may extend to autopolyploids as well (17, 72), and we therefore include autopolyploidy in this review.] Thus, an autotetraploid will contain four ...

Towards a better understanding of polyploid Sorbus (Rosaceae) from Bosnia and Herzegovina (Balkan Peninsula), including description of a novel, tetraploid apomictic species: Polyploid Sorbus From Bosnia and Herzegovina

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