  Citation: Shekhovtsov, S.V.; Shekhovtsova, I.N.; Kosterin, O.E. Genotyping-by-Sequencing Analysis Shows That Siberian Lindens Are Nested within Tilia cordata Mill. Diversity 2022, 14, 256. https:// doi.org/10.3390/d14040256 Academic Editors: Michael Wink and Federico Sebastiani Received: 7 March 2022 Accepted: 28 March 2022 Published: 30 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). diversity Article Genotyping-by-Sequencing Analysis Shows That Siberian Lindens Are Nested within Tilia cordata Mill Sergei V. Shekhovtsov 1,2, * , Irina N. Shekhovtsova 3 and Oleg E. Kosterin 1 1 Institute of Cytology and Genetics SB RAS, 630090 Novosibirsk, Russia; kosterin@bionet.nsc.ru 2 Institute of the Biological Problems of the North FEB RAS, 685000 Magadan, Russia 3 Central Siberian Botanical Garden of the SB RAS, 630090 Novosibirsk, Russia; maklakovain@mail.ru * Correspondence: shekhovtsov@bionet.nsc.ru Abstract: Tilia sibirica and T. nasczokinii are considered to be endemic Siberian linden species. They have very small distributions located hundreds to thousands of kilometers away from other lindens. It is unclear how closely these species are related to the widespread Tilia cordata: according to the current hypotheses, they could be pre-Pleistocene relicts or remnants of the recent continuous range of T. cordata that existed during the Holocene climatic optimum. Earlier studies detected significant differences between T. sibirica, T. nasczokinii, and T. cordata in microsatellite loci, but not in plastid sequences. Here we performed a phylogenetic analysis of several linden species based on GBS data. The obtained GBS sequences were assembled to create phylogenetic trees based on 16,000–294,000 variable sites. We found that T. cordata and the two putative Siberian species formed a monophyletic group. It consisted of three clades: the basal clade containing specimens from the Caucasus, and two sister clades representing populations from the East European Plains+the Urals and Siberia, respectively. Neither of the Siberian species was related to the Far Eastern T. amurensis, as was hypothesized earlier. Our study suggests that the colonization of Europe and Siberia after the Last Glacial Maximum occurred from different glacial refugia. Keywords: linden; T. cordata; T. sibirica; T. nasczokinii; Siberia; GBS 1. Introduction Tilia cordata Mill. has the largest distribution among Eurasian lindens, from the At- lantic coast of Europe to West Siberia (Figure 1). In the east, its distribution becomes fragmented, with small populations scattered across swamps of the West Siberian Plain [1], up to the Novosibirsk [2] and Tomsk [3] Oblasts. These easternmost outposts of T. cordata are separated by ca. 600 km from the linden populations of the foothills of the Altai–Sayan mountain system, which are regarded as two separate species. A relatively big popula- tion from the Gornaya Shoria Mts (Kemerovo Oblast), the so-called Kuzedeevo Linden Island [4–6], was described as Tilia sibirica Fisch. ex Bayer. Numerous small populations from neighboring regions are also believed to belong to this species [7–9]. T. sibirica differs from T. cordata by having leaf blades on flowering shoots with strait or shallowly-cuneate base, not glaucescent beneath, as well as dense pubescence of ovary [10,11]. Two small adjacent populations growing near Krasnoyarsk were described as Tilia nasczokinii Stepanov. They were characterized as having asymmetrical, transversely cordate or basally truncated leaves, lanceolate lobes of stellate stigma, and few-flowered inflorescences [12]. However, many authors do not recognize these two taxa as separate species, because the diagnostic characters are highly variable so the distinctions can be traced only on a series of individu- als. For example, the authors of the fundamental treatise «Woody plants of Asian Russia» considered both Siberian species as synonyms of T. cordata [13]. Several molecular studies attempted to clarify this issue. Logan et al. [14] employed 12 microsatellite loci to compare T. sibirica from the Kuzedeevo Linden Island to three T. cordata populations from Austria, Poland, and West Siberia. T. sibirica was found to differ Diversity 2022, 14, 256. https://doi.org/10.3390/d14040256 https://www.mdpi.com/journal/diversity