Annotation of the results obtained in 2023
1 A study was conducted of the structure of genetic variability of 17 nuclear microsatellite loci and one newly developed mitochondrial marker in fir species from the Balsamea section of the Russian Far East. Using the STRUCTURE method, differentiation of the Sakhalin fir Abies sachalinensis into four groups in the latitudinal direction was revealed (Fig. 1). These groups were distinguished from the continental fir populations of A. nephrolepis and Kamchatka fir A. gracilis. The presence of A. sachalinensis genes has been noted in populations on the western shore of the Strait of Tatar, indicating genetic flow between Sakhalin and the continent. 2 Polymorphism of two SNPs in the mitochondrial genome of Far Eastern firs was confirmed. One of them differentiates Sakhalin fir into a southern and northern cluster, and the presence of the South Sakhalin mitotype on the western side of the Tatar Strait confirms the transfer of seeds across the strait. 3 A demographic study of Far Eastern species based on microsatellite data and using ABC modeling identified the most supported demographic scenario, in which the Kamchatka fir A. gracilis diverged from the North Sakhalin populations rather than from A. nephrolepis or the South Sakhalin A. sachalinensis. According to ABC estimates, the split of the ancestor of the continental A. nephrolepis and the ancestor of the southern form of A. sachalinensis occurred about 600 thousand years ago; A. nephrolepis and the northern form of A. sachalinensis separated around 300 thousand years ago; Kamchatka fir A. gracilis separated from the northern form of A. sachalinensis 37 thousand years ago; the populations of the central part of Sakhalin arose as a result of admixture of the North Sakhalin and South Sakhalin populations 18 thousand years ago; fir in the Kuril Islands and Hokkaido separated from the South Sakhalin populations about 118 thousand years ago. n. 4 ABC study of Siberian fir using 17 microsatellite loci estimated the separation of the Yenisei populations of Siberian fir from the Baikal ones occurred about 28 thousand years ago, and the hybrid origin of the North Ural populations as a result of admixture of the South Ural and Baikal populations about 35 thousand years ago, i.e. around the last glacial maximum. 5 For Siberian pine, 2 multiplex sets of nuclear microsatellite markers (20 loci) have been developed for analysis on a sequencer. 6 It has been established that in the north of the Urals, Siberian pine populations are of mixed origin from the descendants of the populations from the Kuznetsk Alatau and the Middle Urals. According to ABC modeling, the formation of mixed North Ural populations occurred during the early-mid Holocene (about 10 thousand years ago). In addition, the increased number of microsatellite loci (20) and greater sampling of material once again confirmed the previously obtained estimates of the time of separation of the Siberian pine populations of the Urals and Kuznetsk Alatau (37-20 thousand years ago), which coincided with the time of sharp climatic changes in MIS 2 period (marine isotope stage). 7. During the analysis of genetic differentiation using the STRUCTURE method of Siberian pine from the Middle Urals, Northern Urals and Cis-Urals, it was discovered that the populations of the Cis-Urals have a secondary mixed origin from populations of the Middle Urals and “admixture” populations of the Northern Urals. Also, according to ABC estimates, the age of the “admixture” event for the westernmost population of pine in the Cis-Urals is estimated at 4 thousand years ago. 8. The hypothesis about the presence of a Yenisei cluster of populations in the Siberian pine, similar to that of the Siberian fir, was not confirmed. Siberian pine, growing on the Yenisei Plain and in neighboring geographical areas (Chulym-Yenisei Basin, Minusinsk Basin, Chulym Plain), has a mixed origin from clusters of populations of the Western Sayan and Kuznetsk Alatau. 9. Checking the hybridogenic status of the origin of Siberian pine populations from Western Sayan using population structure analysis and modeling demographic scenarios as descendants of Altai and Eastern Sayan populations established that a group of pine populations from Western Sayan descended from Altai populations approximately 8 thousand years ago. Thus, the mixed status of pine pine populations from the Western Sayan was not confirmed. However, the pine populations of the Western Sayan subsequently experienced, in the Holocene, the influence of gene flow from the pine populations of the Eastern Sayan, increasing to the east. 10. The results of research within the framework of this RSF project are posted on the network: https://ipae.uran.ru/node/1295

 

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Annotation of the results obtained in 2022
Studies of the population structure and genetic variability of northern Eurasian firs, carried out using allozyme markers, cpSSRs, AFLP, and mitochondrial DNA, have expanded our understanding of the origin and history of these species. Nevertheless, the data obtained were clearly insufficient to judge with certainty about past demographic processes within species in their connection with Pleistocene climatic oscillations and, as a result, changes in the paleogeographic situation in the territory of Northern Eurasia. For another important component of the dark coniferous taiga formation, the Siberian pine, no data that would allow drawing conclusions about its historical biogeography have yet been presented. Modeling demographic events of the past (changes in population size, time of divergence-hybridization of populations, etc.) requires the use of unlinked autosomal loci with a sufficiently high level of variability. Therefore, we used nuclear microsatellite markers, which are well suited for analyzing genetic variation and establishing genetic relationships between populations (Selkoe and Toonen 2006). In this project, we investigate the demographic models of Siberian fir, Semenov fir, and Siberian pine by estimating the parameters of past demographic events based on data on the variability of nuclear microsatellite loci. The construction of demographic models of the considered species was based on a number of prerequisites. To reveal the ancient migration history, we used data on the variability of mitochondrial DNA loci. Information on past species ranges was obtained from a survey of pollen and macrofossil databases (Binney et al. 2009; 2017). We also applied the maximum entropy (MaxEnt) method to model the distribution of species in past climatic periods. Using these approaches, a number of demographic history models were formally validated to determine the most likely scenarios and parameters of demographic models, such as the age of key divergence-hybridization events, effective population size, hybridization parameters, etc. The following results were obtained: 1. 1135 individuals of five species of fir were genotyped based on 15-18 nuclear microsatellite loci, depending on the species. 2. Estimates of intraspecific variability have been obtained. In Siberian fir, the average number of alleles (Na), observed and expected heterozygosities (Ho and He) were 5.212, 0.565, and 0.569, respectively. In Semenov fir, these parameters were much smaller: 1.600, 0.054, and 0.097, respectively, which obviously reflects the long-term isolation of this species from Siberian fir and the small size of the population. 3. The study of the structure of variability of Siberian fir using the STRUCTURE method with the number of groups K = 5 revealed the following groups: Altai, Salair and Kuznetsk Alatau (i), Southern and Middle Urals (ii), Northern and Subpolar Urals (iii), Baikal and Eastern Sayan (iv), Lower Yenisei (v) (Fig. 1). 4. As a result of demographic modeling of Siberian fir populations based on genotypic data on nuclear microsatellite loci, the most likely scenario was chosen (includes the separation of the Altai and Baikal groups with further separation from the Baikal group of the South Urals and finally the emergence of the North Ural group as a result of admixure of Baikal and South Ural groups, Fig. 2) and the posterior distributions of the model parameters were calculated: the effective population size of the groups (median) varied from 3000 in the Northern Urals to 15000 in Altai. The time of formation of the North Ural group (median) was 34,000 years, assuming a generation time of 100 years, and a hybridization coefficient r1 = 0.8, meaning that the Northern Urals was mainly populated by migrants from the Southern Urals and, to a lesser extent, by migrants from the Baikal region. The southern Urals population arose as a result of settlement from the Baikal region about 90,000 years ago, and the Altai and Baikal groups diverged about 165,000 years ago. The contribution of the Altai group to the genetic pool of the South Ural group, if we consider an alternative scenario with a hybrid origin of the latter, turns out to be less than 0.1. This looks strange if we take into account the presence of the Altai mitotype in the south of the Urals. Perhaps this discrepancy is due to the fact that during migrations in the source of colonization in Southern Siberia, both mitotypes, now segregated in the Altai-Western Sayan region and in the Eastern Sayan-Baikal region, respectively, coexisted together and penetrated to the Urals together. It is also possible that the colonization of the Southern Urals occurred both from the Altai group and from the Baikal group, but not simultaneously, and the adaptation of these groups to different ecological conditions determined the selective advantage of the Baikal group, which basically replaced the genes of the Altai group in the Urals in the process of hybridization. At the same time, the Altai mitotype was preserved due to maternal inheritance of mitochondrial DNA. 6. Modeling the demography of Semenov fir gave estimates of the effective population size of Semenov fir and two South Siberian groups of Siberian fir (Altai and Baikal) as 200 individuals, 15,000 and 10,000 individuals, respectively. The time of separation of the Semenov and Siberian firs is estimated as 1.0 – 2.5 Ma. These estimates explain the extremely low level of variability of Semenov fir. They are quite consistent with the existing ideas about the steady trend towards aridization of Central Asia throughout the Pleistocene, due to both global climatic processes and regional factors - the continued growth of the mountain ranges of Central Asia - the Himalayas, Hindu Kush, Kun-Lun, Tien Shan (Fortuna et al., 2018; Song et al., 2021). 7. Siberian stone pine (Pinus sibirica Du Tour) is one of the key components of the dark coniferous taiga formation, the biogeographic history of which has remained poorly understood until recently. In this study, we carried out a comprehensive reconstruction of the late Quaternary history of Siberian pine. To do this, we used data from genetic variability, paleobotany, and ecological niche modeling to penetrate into the past of the species and identify refugia and dispersal patterns of Siberian pine from the last interglacial to the Holocene. In total, 42 natural populations of Siberian pine (1130 trees) were studied, representing a large part of the species range. The mitochondrial pattern of variation was revealed using two loci by single-stranded conformational polymorphism (SSCP). Demographic scenarios and population structure were examined using 8 nuclear microsatellite DNA loci. For the paleobotanical review, we used published works on pollen and macrofossils of Siberian pine using the database of Binney et al. (2009; 2017). Ecological modeling of the range of Siberian pine was carried out using the MaxEnt 3.4.1 program for the present, the Middle Holocene (8-4 ka), the last glacial maximum (LGM) (21 ka) and the last interglacial (130 t . l. n.). During the study, it was found that the populations of Siberian pine from the Western Sayan and Altai, Eastern Sayan, Kuznetsk Alatau and the Urals represent four genetically isolated clusters (Fig. 3, 4), preserved in mountain refugia during the last glacial maximum (22 thousand years ago). However, the results of the analysis of the variability of mitochondrial and nuclear DNA showed that the processes of the spread of Siberian pine in the subsequent Holocene epoch (last 10000 years) were heterogeneous. Thus, Siberian stone pine populations from Altai and Western Sayan did not participate in the recolonization of the West Siberian Plain, since they had a common mitotype, which was not found outside the Altai-Sayan mountainous country. Eastern populations of Siberian pine dispersed in the Holocene from the Eastern Sayan and / or the coast of Lake. Baikal. In the Holocene, Siberian pine populated the West Siberian Plain from two centers – the Urals and the Kuznetsk Alatau. At the same time, two groups of populations were formed, separated approximately along the middle of the West Siberian Plain. However, the populations of the Urals and Kuznetsk Alatau have a common mitotype and probably diverged relatively recently during the early Zyryanka glaciation (71-57 thousand years ago) and/or Sartan glaciation (29-14 thousand years ago). To estimate the time of divergence, ABC modeling was carried out on the basis of microsatellite data which regards the demographic scenarios for the separation of the Ural group and the Kuznetsk Alatau group. One demographic scenario was tested (Fig. 5), where: t2 is the time of divergence of the populations in Southern Siberia (Altai, Kuznetsk Alatau, Eastern Sayan); t1 is the time of separation of the Ural group of populations from the Kuznetsk Alatau group. The ABC simulations determined the distribution mode of the time of this divergence to be approximately 37.8 kyr with a confidence interval of 10-100 thousand y. a. 37.8 thousand y. a. corresponds to the end of Karginskii interstadial and the beginning of the Sartan glaciation, but the confidence interval did not exclude the possibility of the first divergence of the Ural group during the early Zyryanka glaciation. P. sibirica could have penetrated into the Urals from the Kuznetsk Alatau during the last interglacial (Kazantsevo: 130-71 thousand years ago). Ecological niche modeling and paleodata indicated favorable conditions for Siberian pine in Western Siberia during the Kazantsevo Interglacial. Lately, two disjunctions between the Urals and the Kuznetsk Alatau could have occurred during two successive glaciations - Early Zyryanka (71-57 thousand years ago) and Sartan (29-14 thousand years ago).

 

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