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Pleistocene range disruption and postglacial expansion with secondary contacts explain the genetic and cytotype structure in the western Balkan endemic Alyssum austrodalmaticum (Brassicaceae)

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Abstract

The western Balkans represents an area of significant topographic and environmental heterogeneity, harbouring high species and intraspecific diversity. Similar genetic and distributional splits observed in unrelated species have suggested some common features in their glacial response and biogeographic history. Here, we studied the western Balkan endemic Alyssum austrodalmaticum with the aim to explore and understand its intraspecific structure and processes that shaped the present patterns. We employed data from AFLPs, two low-copy nuclear genes, genome size, morphometrics and species distribution modelling. Four genetic lineages were identified within the species, which were geographically structured and showed cytotype-specific patterns. The observed phylogeographic structure is congruent with the predicted species distribution during the Last Glacial Maximum. Two allopatric diploid lineages (referred to as N2x and S2x) reflect glacial range disruption and survival in distinct refugia inferred in north-western (Istria, Kvarner) and south-eastern Adriatic areas (northern Adriatic palaeo-coastline and southern Dinarides). AFLP analyses with in silico-generated polyploid genotypes and nuclear genes proved that the two tetraploid lineages (C4x, S4x) were genetic allopolyploids and arose independently. The central tetraploids (C4x) originated through secondary contacts of the two diploid lineages. The origin of the southern tetraploids (S4x) is somewhat ambiguous. Apart from the southern diploids (S2x), the involvement, either direct or through later introgression, of the central tetraploids (C4x) or even other Balkan relatives is possible. Our study highlights the role of glacial range dynamics and secondary contacts, triggering introgression and polyploid evolution, in the formation of genetic diversity and intraspecific patterns in the western Balkans.

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Acknowledgements

This study was financially supported by the Slovak Research and Development Agency (APVV; Grant No. APVV-17-0616) and by the GAČR—Czech Science Foundation (Grant No. 19-06632S). We are grateful to M. Perný and J. Šibík (Plant Science and Biodiversity Centre, SAS, Bratislava, Slovakia), who accompanied us in the field; D. Brišková (Berlin-Chemie AG, Bratislava, Slovakia), who generated DET1 sequence data for her diploma thesis and kindly provided them for the present study; I. Rešetnik (University of Zagreb, Croatia) for extracting and providing distribution data from the Flora Croatica Database; and J. Eriksson (University of Gothenburg, Sweden) for her advice on assembling the input file for AlloppNET computations. We also wish to thank two reviewers, who provided valuable comments and helped to improve the manuscript.

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Correspondence to Judita Zozomová-Lihová.

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Handling Editor: Ivana Rešetnik.

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Contribution to “Plants of the Balkan Peninsula in Space and Time”.

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Appendices

Appendix

List of population samples of Alyssum austrodalmaticum and related species under study. Populations of A. austrodalmaticum were subjected to morphometric and relative genome size measurements, and to AFLP and nuclear gene sequence analyses. The other listed species were included only in analyses of nuclear genes. The locality description and collection data are followed by the number of individuals included in AFLP, morphological (morph) and genome size (FCM-gs) analyses (if applicable), and by GenBank accession numbers for the DET1 and CHS genes (if applicable). GenBank accession numbers with superscripts refer to sequences generated in earlier studies: Melichárková et al. (2017a, 2019b); otherwise, the sequences were generated in the present study. Population codes (in bold) are identical to the collection numbers. All voucher specimens were deposited in the herbarium of the Institute of Botany, SAS, Bratislava, Slovakia (SAV).


Alyssum austrodalmaticum Trinajstić: northern diploids (lineage N2x).


53KAS, Slovenia, Primorska, Submediteransko območje, NW of the village of Kastelec, karst grassland near the cemetery, 45° 35.017′ N, 13° 51.699′ E, 357 m a. s. l., 27 May 2007, S. Španiel and M. Perný; AFLP—7 ind., morph—13 ind., FCM-gs—9 ind., DET1: MN685397–MN685399; CHS: MN685498–MN685503. 97SKR, Croatia, Primorsko-goranska županija, Krk Island, Skrbčići, along the road S of the village, 45° 02.45′ N, 14° 29.6′ E, 78 m a. s. l., 17 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—20 ind., FCM-gs—10 ind., DET1: MN685439–MN685442; CHS: MN685556–MN685561. 98OSO, Croatia, Primorsko-goranska županija, Cres Island, Osor, along the road NE of the village, 44° 41.9′ N, 14° 23.883′ E, 28 m a. s. l., 17 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—20 ind., FCM-gs—10 ind., DET1: MN685419–MN685422; CHS: MN685530–MN685534. 99CRE, Croatia, Primorsko-goranska županija, Cres Island, along the road N of the town of Cres, 44° 58.95′ N, 14° 24.667′ E, 157 m a. s. l., 17 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—20 ind., FCM-gs—10 ind. 100BKAa,b, Croatia, Primorsko-goranska županija, Krk Island, Baška valley, two microlocalities—45° 01.683′ N, 14° 40.633′ E, 273 m a. s. l. and 44° 58.133′ N, 14° 46.017′ E, 41 m a. s. l., 17 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—33 ind., FCM-gs—17 ind. 101GRO, Croatia, Primorsko-goranska županija, town of Grobnik, Grobničko polje, grassland near the motor-racing circuit, 45° 23.000′ N, 14° 31.046′ E, 318 m a. s. l., 18 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—23 ind., FCM-gs—9 ind., DET1: MN685381–MN685386; CHS: MN685483–MN685485. 104RAB, Croatia, Primorsko-goranska županija, Rab Island, rocky area near the port of Mišnjak, 44° 42.417′ N, 14° 51.583′ E, 10 m a. s. l., 18 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—29 ind., FCM-gs—10 ind., DET1: MN685433–MN685436; CHS: MN685551–MN685554. 105KAG, Croatia, Ličko-senjska županija, S of the town of Karlobag, rocks near the road along the coast, 44° 31.05′ N, 15° 05.333′ E, 25 m a. s. l., 18 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—28 ind., FCM-gs—9 ind. 106PAG, Croatia, Zadarska županija, Pag Island, rocks near the road above the town of Pag, 44° 26.333′ N, 15° 02.233′ E, 162 m a. s. l., 19 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—30 ind., FCM-gs—10 ind., DET1: MN685423-MN685427; CHS: MN685535-MN685540. 107OBR, Croatia, Zadarska županija, S of the town of Obrovać, near the crossroad Knin-Zadar-Obrovać, 44° 11.783′ N, 15° 40.85′ E, 98 m a. s. l., 19 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—24 ind., FCM-gs—9 ind., DET1: MN685413–MN685418; CHS: MN685524–MN685529. 108PJS, Croatia, Šibensko-kninska županija, N of Knin, near Prevjes, between villages of Padene and Palanka, 44° 06.733′ N, 16° 04.65′ E, 338 m a. s. l., 19 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—22 ind., FCM-gs—10 ind. 126KNI, Croatia, Šibensko-kninska županija, town of Knin, grazing area near the road, 44° 03.05′ N, 16° 11.15′ E, 327 m a. s. l., 26 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—24 ind., FCM-gs—10 ind., DET1: MN685400–MN685403; CHS: MN685506–MN685511. 127LJU-diploids, Croatia, Ličko-senjska županija, the village of Ljubovo, grazing land and rocks (minefield), 44° 39.667′ N, 15° 33.433′ E, 935 m a. s. l., 28 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—4 ind., FCM-gs—5 ind. 128BRS, Croatia, Primorsko-goranska županija, W of the village of Brseč (ca 13 km S of Lovran), rocky slope and the summit area of Mt. Sisol, 45° 10.6′ N, 14° 12.2′ E, 764 m a. s. l., 27 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—0 ind., FCM-gs—10 ind. 129OCI, Slovenia, Primorska, between villages of Ocizla and Petrinje, ruderal site near the road, 45° 34′30″ N, 13° 54′39″ E, 411 m a. s. l., 28 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—0 ind., FCM-gs—10 ind. 130LOR, Italy, Friuli-Venezia Giulia, SE of Basovizza near Trieste, ca 3 km from village of San Lorenzo, rocks and hayfield near the road, 45° 37′46″ N 13° 53′34″ E, 482 m a. s. l., 28 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—0 ind., FCM-gs—10 ind. 131OPI, Italy, Friuli-Venezia Giulia, the village of Opicina near the town of Trieste, grassland near the road, 45° 41.633′ N, 13° 48.85′ E, 318 m a. s. l., 28 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—22 ind., FCM-gs—10 ind.


Alyssum austrodalmaticum: central tetraploids (lineage C4x).


127LJU-tetraploids, Croatia, Ličko-senjska županija, the village of Ljubovo, grazing land and rocks (minefield), 44° 39.667′ N, 15° 33.433′ E, 935 m a. s. l., 28 Apr 2008, S. Španiel and M. Perný; AFLP—0 ind., morph—6 ind., FCM-gs—23 ind. 124DRR, Bosnia and Herzegovina, Kanton 10 (Livanjski kanton), rocky face near the road between the village of Drvar and the settlement of Resanovci, 44° 20.85′ N, 16° 21.117′ E, 750 m a. s. l., 26 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—0 ind., FCM-gs—10 ind., DET1: MN685369–MN685380; CHS: MN685472–MN685482. 240HUM, Croatia, Splitsko-dalmatinska županija, Brač Island, along the road NW of the settlement of Gornji Humac, 43° 16.497′ N, 16° 42.623′ E, 435 m a. s. l., 16 May 2009, S. Španiel and J. Šibík; AFLP—7 ind., morph—21 ind., FCM-gs—10 ind., DET1: MN685387–MN685396; CHS: MN685486–MN685497. 241CAA, Croatia, Dubrovačko-neretvanska županija, Korčula Island, along the road between the villages of Čara and Pupnat, 42° 56.395′ N, 16° 57.132′ E, 284 m a. s. l., 17 May 2009, S. Španiel and J. Šibík; AFLP—6 ind., morph—18 ind., FCM-gs—10 ind., DET1: MN685360–MN685368; CHS: MN685460–MN685471.


Alyssum austrodalmaticum: southern diploids (lineage S2x).


111NEU, Croatia, Dubrovačko-neretvanska županija, SE of the town of Neum, rock face along the road near the shore, 1.5 km S of the border Bosnia and Herzegovina—Croatia, 42° 52.917′ N, 17° 39.75′ E, 60 m a. s. l., 20 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—30 ind., FCM-gs—10 ind., DET1: MN685407–MN685412; CHS: MN685518–MN685523. 117PON, Croatia, Dubrovačko-neretvanska županija, Pelješac Peninsula, rocks near the road in the village of Ponikve (Boljenovići), 42° 50.933′ N, 17° 37.317′ E, 223 m a. s. l., 23 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—30 ind., FCM-gs—10 ind., DET1: MN685429–MN685432; CHS: MN685544–MN685548. 119KOR, Croatia, Dubrovačko-neretvanska županija, Mljet Island, between the villages of Korita and Maranovići, 42° 42.6′ N, 17° 41.267′ E, 250 m a. s. l., 23 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—30 ind., FCM-gs—10 ind., DET1: MN685404–MN685406; CHS: MN685512–MN685515.


Alyssum austrodalmaticum: southern tetraploids (lineage S4x).


116SRD, Croatia, Dubrovačko-neretvanska županija, Mt. Srd above the town of Dubrovnik, 42° 38.85′ N, 18° 06.95′ E, 348 m a. s. l., 23 Apr 2008, S. Španiel and M. Perný; AFLP—7 ind., morph—29 ind., FCM-gs—11 ind., DET1: MN685443–MN685454; CHS: MN685562–MN685571.


Alyssum bosniacum Beck.


199BOR, Bosnia and Herzegovina, Hercegovačko-neretvanski kanton, Prenj Mts, Mt. Borašnica, W of the village of Borci (15 km S of the town of Konjic), 43° 33.902′ N, 17° 57.695′ E, 1590 m a. s. l., 22 Jun 2008, S. Španiel and M. Perný; DET1: MK164705b; CHS: MN685458, MN685459. 200PRO, Bosnia and Herzegovina, Srednjobosanski kanton, Vranica Mts, W of the town of Fojnica, pasture above Prokoško jezero lake, 43° 56.912′ N, 17° 45.370′ E, 1808 m a. s. l., 23 Jun 2008, S. Španiel and M. Perný; DET1: MK164805b; CHS: MN685549, MN685550.


Alyssum gmelinii Jord. & Fourr.


52KRA, Austria, Steiermark, Kraubath an der Mur, serpentine rocks in the pine forest, 47° 16.93′ N, 14° 55.65′ E, 635 m, 26 May 2007, S. Španiel and M. Perný; DET1: KX697250a; CHS: KX697101a. 133KEL, Serbia, Severna Bačka, Kelebija near the town of Subotica, 46° 09.154′ N, 19° 38.627′ E, 129 m, 13 May 2008, S. Španiel and J. Šibík; DET1: KX697247a; CHS: KX697098a.


Alyssum moellendorfianum Asch. ex Beck.


120KIC, Bosnia and Herzegovina, Hercegovačko-neretvanski kanton, town of Konjic, limestone rocks and screes, 43° 39.85′ N, 17° 58.267′ E, 370 m a. s. l., 25 Apr 2008, S. Španiel and M. Perný; DET1: MK164739–MK164740b; CHS: MN685504, MN685505. 121POD, Bosnia and Herzegovina, Hercegovačko-neretvanski kanton, village of Podorašac, limestone rocks and screes, 43° 41.583′ N, 17° 59.1′ E, 360 m a. s. l., 25 Apr 2008, S. Španiel and J. Šibík; DET1: MK164795b; CHS: MN685543.


Alyssum montenegrinum (Bald.) Španiel, Lihová & Marhold.


115PET, Montenegro, Budva, rock face along the road near the town of Petrovac (towards the town of Virpazar), 42° 13.028′ N, 18° 57.334′ E, 392 m a. s. l., 22 Apr 2008, S. Španiel and M. Perný; DET1: MN685428; CHS: MN685541–MN685542. 192BOA, Montenegro, Šavnik, between the settlements of Šavnik and Boan near a crossroad to Timar, along the road above the river Bukovica, limestone screes, 42° 59.202′ N, 19° 11.302′ E, 1193 m a. s. l., 19 Jun 2008, S. Španiel and M. Perný; DET1: MN685358, MN685359; CHS: MN685456, MN685457.


Alyssum repens Baumg.


419JHN, Austria, Kärnten, SE of Sankt Paul im Lavanttal, Johannesberg, grasslands near the church in the village, 46° 41.633′ N, 14° 52.733′ E, 606 m a. s. l., 14 May 2013, W. Franz, A. Drescher, S. Španiel, J. Zozomová-Lihová and K. Marhold; DET1: KX697246a; CHS: KX697097a. 416PEG, Austria, Steiermark, Steirisches Randgebirge, Murtal N Peggau, northern part of the Peggauer Wand, rock face, 47° 12.550′ N, 15° 20.816′ E, 522 m a. s. l., 13 May 2013, A. Drescher, S. Španiel, J. Zozomová-Lihová and K. Marhold; DET1: KX697264–KX697265a; CHS: KX697115–KX697116a.


Alyssum spruneri Jord. & Fourr. subgroup ‘serbicum’ (for its circumscription, see Španiel et al. 2017a, b).


140TOP, Serbia, Pirot, the village of Topli do (NE of the town of Pirot) ruderal sites and rocks along the road, 43° 17.407′ N, 22° 35.283′ E, 446 m a. s. l., 17 May 2008, S. Španiel and J. Šibík; DET1: MK164830–MK164831b; CHS: MN685573. 143MAG, Serbia, Raška, Maglič fortress above the river Ibar, serpentine slopes, 43° 36.782′ N, 20° 33.065′ E, 374 m a. s. l., 18 May 2008, S. Španiel and J. Šibík; DET1: MK164759b; CHS: MN685516, MN685517.


Alyssum vernale Kit. ex Hornem.


27SKE, Romania, Mehedinţi, Schela Cladovei (part of the town of Drobeta-Turnu Severin, 3 km W of the town), 44° 38.065′ N, 22° 35.610′ E, 70 m a. s. l., 5 May 2007, S. Španiel, J. Zozomová-Lihová, K. Marhold and V. Kolarčik [The locality was recently destroyed due to the construction of a road.]; DET1: MN685437, MN685438; CHS: MN685555. 134SUM, Serbia, Južni Banat, Deliblatska peščara, the village of Šumarak, between the villages of Gaj and Dubovac, 44° 49.106′ N, 21° 08.581′ E, 105 m a. s. l., 13 May 2008, S. Španiel and J. Šibík; DET1: MN685455; CHS: MN685572.

Information on electronic supplementary material

Online Resource 1. Map showing distribution records of Alyssum austrodalmaticum used for species distribution modelling, gathered from the here sampled populations and from the data stored in the Flora Croatica Database (Nikolić 2019, available at: https://hirc.botanic.hr/fcd).

Online Resource 2. Morphological characters used in the present study, and the results of morphometric analyses (eigenvectors of PCA, total canonical structure of CDA).

Online Resource 3. Cohen’s d and its 95% confidence interval of Nei-Li genetic distances between in silico tetraploids inferred under different scenarios and natural sampled tetraploids of Alyssum austrodalmaticum (lineages C4x and S4x) based on AFLP data.

Online Resource 4. Bayesian majority-rule consensus tree based on CHS sequence data obtained from Alyssum austrodalmaticum and its close relatives.

Online Resource 5. Summary data of flow cytometric measurements of relative genome size in Alyssum austrodalmaticum and its genetic lineages.

Online Resource 6. Principal component analysis of Alyssum austrodalmaticum at the level of individual plants based on 20 morphological characters.

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Zozomová-Lihová, J., Melichárková, A., Svitok, M. et al. Pleistocene range disruption and postglacial expansion with secondary contacts explain the genetic and cytotype structure in the western Balkan endemic Alyssum austrodalmaticum (Brassicaceae). Plant Syst Evol 306, 47 (2020). https://doi.org/10.1007/s00606-020-01677-5

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