Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: A revised view of the taxonomic treatment of the Ojców birch.

Birches are generally known for their high genetic and morphological variability, which has resulted in the description of many species. Ojców birch was described in 1809 by Willibald Suibert Joseph Gottlieb Besser in Poland. Since then, several studies assessing its taxonomy were conducted. Today, various authors present Ojców birch at different taxonomic ranks. In Czechia, the Ojców birch is classified a critically endangered taxon and confirmed at one locality consisting of several tens of individuals. However, before a strategy for its conservation can be applied, we consider it necessary to assess the taxonomic position of the endangered Czech population and to evaluate its relationship to the original Polish population. This study aimed to evaluate the morphometric and genetic variability between populations of B. ×oycoviensis in Poland and the Czechia and their relationship to regional populations of B. pendula, one of the putative parental species of the Ojców birch. Altogether, 106 individuals were sampled, including the holotype of B. szaferi, the second putative parental species of B. ×oycoviensis, received from the herbarium of W. Szafer, which is deposited at the Institute of Botany in Kraków. Morphological analyses identified differences in leaves between B. ×oycoviensis and B. pendula. However, no significant differences were found in genome size between selected taxa/working units except for B. pendula sampled in Czechia. The identified difference of the Czech population of B. pendula is probably caused by population variability. Genetic variability between all the taxa under comparison, regardless of their origin, was also very low; only the benchmark taxa (B. nana and B. humilis) clearly differed from all samples analyzed. The results indicate minute morphological and negligible genetic variability between the Czech and Polish populations of B. ×oycoviensis. In light of our results, the classification of B. ×oycoviensis as B. pendula var. oycoviensis seems more accurate than all hitherto presented alternatives (e.g. B. ×oycoviensis as a separate species).

Introduction

The genus Betula is generally known for its extensive genetic and morphological variability among taxa [1, 2]. The main reasons for such variation are frequent hybridization and subsequent introgression as well as polyploidization [3-5]. As a consequence, ca 64 birch species are currently distinguished worldwide [2]. However, the taxonomic treatment of some birches is ambiguous, and various authors present different numbers of species, usually ranging between 30 and 60 [6, 7]. Several minute birch taxa with questionable taxonomic position were also described in Central Europe in the 19th and 20th centuries, such as B. carpatica, B. obscura, B. atrata, and B. petraea [8-11]. One intricate taxon of the genus Betula is the Ojców birch (Betula ×oycoviensis Besser), which was first described by Besser [12] as B. oycoviensis. Ojców birch is a diploid taxon (2n = 28) closely related to silver birch (B. pendula). The taxonomic position of the Ojców birch and the character of its relationship to silver birch, however, remains unresolved. A detailed description of the taxon was made available in 1921 [13], and the species B. oycoviensis was recognized further in 1928, based on the description of samples taken in Hamernia (Poland) [14]. Since then, several studies focused on determination its origin have been conducted [15-18]. Based on this subsequent research, B. oycoviensis was classified as a hybrid between B. pendula and ‘Betula nova’ [15]. ‘Betula nova’ was later described as Betula szaferi Jentys-Szaferowa ex Staszkiewicz [19]. Throughout the following text, we refer to Oyców birch using the established scientific name B. ×oycoviensis (as did, for example, Rutkowski [20]), although some authors have recently classified this birch at lower taxonomic ranks, for example as B. pendula var. oycoviensis [21].

Betula ×oycoviensis is in many traits similar to B. pendula. The most prominent differences can be observed on its leaves, which often grow in groups of 4–6 on brachyblasts and are up to 4 cm long, and in its habitus: Compared to B. pendula, B. ×oycoviensis generally has the form of lower trees (up to 15 m) or shrubs [17, 20–22] with a curved stem and ‘broomy’ crown. Betula ×oycoviensis has no continuous area of distribution. Besides Poland, some isolated micropopulations of Betula ×oycoviensis occur or probably occur in Czechia, Romania, Ukraine, Denmark and Sweden [22-25].

In Poland, several places of occurrence have been reported, for example Dolina Kobylańska (close to Kraków, Lesser Poland Voivodeship), Skielek (Beskid Wyspowy, Lesser Poland Voivodeship) and Czerwona Góra (close to Opatów, Świętokrzyskie Voivodeship) [16, 19, 20]. The number of these places has possibly decreased over the past several decades. The fate of a small group of specimens at Chojnik is not known at present, see the updated version (2013) of a study by Staszkiewicz [26], available at http://archive.is/43WK. In Czechia, there is only one confirmed locality of B. ×oycoviensis at the village Volyně u Výsluní in the Ore Mts, North Bohemia [27] and a few proposed unconfirmed localities. Besides the Ore Mts., the Database of the Czech flora and vegetation PLADIAS (https://pladias.cz/en/ to date of 2020/04/07) suggests other possible occurrences of B. ×oycoviensis elsewhere in the country—in the Křivoklátsko region (Central Bohemia) and the Třeboňsko area (South Bohemia), see Fig 1.

Fig 1

Distribution of sampling locations along with other reported locations of B. ×oycoviensis.

Sample locations for additional cytometric analyses (see the Discussion) are depicted using grey circles. Locations with the acknowledged (generally accepted) occurrence of B. ×oycoviensis are marked with filled red triangles (or red dots inside of black circles, if these locations were sampled within our study). Empty red triangles depict locations of proposed (unverified) occurrence of B. ×oycoviensis according to Database of the Czech flora and vegetation PLADIAS (https://pladias.cz/en/ to date of 2020/04/07) and study by Staszkiewicz [18].

Some studies assessing the origin of B. ×oycoviensis have already been published (e.g. the already mentioned studies by Jentys-Szaferowa [28] or Staszkiewicz [19]). However, detailed studies comparing the genetic variation of B. ×oycoviensis and B. pendula populations in Central Europe are missing.

This study aims to assess the morphological and genetic variability between Czech and Polish populations of B. ×oycoviensis and to compare these populations with local populations of B. pendula. The Polish populations of B. ×oycoviensis were selected for our comparison because the original locations from which B. ×oycoviensis was described are in Poland. Moreover, B. ×oycoviensis is often reported to be of hybrid origin [15]. One of its proposed parental species, B. szaferi, was available to us (original acknowledged specimen—holotype) only as a herbarium item from the collections in Kraków (Poland), see Material and methods.

Besides the taxonomic perspective, such studies are also desirable from a practical standpoint. Management steps should be taken towards the effective conservation of the Czech Ojców birch population because it is growing old and its natural regeneration is poor. However, these steps should reflect the taxonomical position of the Ojców birch, so it is important to determine whether its Czech population is taxonomically identical to that in Poland. If it is, Polish individuals could theoretically be used to strengthen the Czech population.

For assessing the variability between B. ×oycoviensis and closely related B. pendula in Poland and Czechia, we employed leaf morphometry, genome size analysis and microsatellite analysis. Previously, leaf morphometry was employed for the study of relationships between birch taxa, for example by Gardiner et al. [29], Atkinson [30] or Gill and Davy [31], whose studies served as a basis for the selection of parameters used in this study. The presented microsatellite analysis was recently used for the determination of taxonomic relationships of the Carpathian birch [32] and was selected based on studies by Kulju et al. [33] and Tsuda et al. [34, 35].

Material and methods

Plant material

Individuals of B. ×oycoviensis for our study were sampled between 2017 and 2019 at Dolina Kobylańska and Skielek in Poland and at Volyně u Výsluní (in the Ore Mts) in Czechia. The specimens of B. ×oycoviensis were complemented by samples taken in the Botanical Garden of the Jagiellonian University (BGJU) in Kraków (Poland) and at Chomutov ZOO (Czechia; the sampled trees were transplanted from Volyně in the past). One birch showing several traits of B. ×oycoviensis was found and sampled in the Křivoklátsko region (Czechia). Reference samples of B. pendula originate from the localities Božídarské rašeliniště, Jezerka, Novodomské rašeliniště and Úhošť in the Ore Mts. (Czechia), and Skielek (Poland). The Czech samples of B. pendula, together with a sample of B. nana described below, were collected in 2012 and used in our previous study [32]. As benchmark species for the evaluation, we included two additional birch taxa (B. nana, also collected in 2012 at Božídarské rašeliniště in the Ore Mts., Czechia, ca. 24 km to the west of the town of Volyně, and B. humilis, which was provided by the BGJU). The exact location (using Leica GPS 1200 at Volyně and Garmin GPSMAP 62S in other cases) and assignment of each sampled individual was recorded in the field. Specific individuals showing some traits of B. szaferi were cultivated at the Research Station Truba near the town of Kostelec nad Černými lesy in Czechia (‘cult. B. szaferi’ for further reference). The ancestors of the ‘cultivated B. szaferi’ specimens from Truba were individuals of B. ×oycoviensis growing at Volyně u Výsluní, whose seeds were collected in 2017. One dry leaf of the holotype (KRAM 303846) of B. szaferi [19] was provided by the herbarium of the W. Szafer Institute of Botany of the Polish Academy of Science (PAS) in Kraków (its photo is included as S1 Fig).

The determination of the taxa was done in the field as follows: As B. ×oycoviensis we determined individuals that exhibited at least 80% of the traits described in the relevant literature [16, 19], and the rest were determined as B. pendula. All other transitional individuals, which combined traits of the two taxa, were determined as B. pendula/B. ×oycoviensis (a ‘working unit’ sensu Kuneš et al. [32]). Besides the benchmark species B. nana and B. humilis, we also collected sample material from B. atrata and B. obscura, which are visually distinct dark-barked birches occurring at the sites of sampling (conducted in 2019), although the taxonomic significance of these dark-barked birches remains questioned. We determined these taxa on the basis of morphological traits described by Domin [10], Hejtmánek [36] and Franiel [37]. Individuals of cult. B. szaferi were classified according to traits referred by Staszkiewicz [19]. However, in contrast to the holotype specimen of B. szaferi from the herbarium in Kraków, the individuals of ‘cult. B. szaferi’ grown at Truba should be viewed more as a working unit than as an acknowledged species. The determination of the remaining taxa (B. nana and B. humilis) is straightforward. From most of the sampled tree individuals, we took three short branches (two for morphometric analysis and one for flow cytometry and molecular analyses).

In total, we sampled twelve taxa and working units from twelve localities (Table 1, Fig 1). We provided a simple table with basic description of all involved taxa in this study as (S1 Table). To simplify the text, we use the term ‘group’ to refer to each taxon or working unit with the country of origin. The limited number of specimens of B. ×oycoviensis in the study is given by the fact that the wild Czech and Polish populations are small. Moreover, the individuals with sufficiently manifested traits of Ojców birch are scattered in stands of B. pendula and other species, and their proportion is low. Even in BGJU in Kraków, only a few trees retained traits of the Ojców birch to such an extent that it allowed us to classify them as unequivocally identifiable B. ×oycoviensis.

Table 1

Numbers of individuals sampled at individual localities in Czechia and Poland.

State	Region	Locality	GPS Coordinates (WGS84)		Group (taxon/working unit)	Number of sampled individuals
			Latitude	Longitude
Czechia	Ore Mts. and surroundings	Božídarské rašeliniště	50.408	12.908	B. nana	1
					B. pendula (CZ)	1
		Chomutov1	50.478	13.431	B. ×oycoviensis (CZ)	2
		Jezerka	50.546	13.481	B. pendula (CZ)	9
		Novodvorské rašeliniště	50.551	13.277	B. pendula (CZ)	2
		Úhošť	50.363	13.239	B. pendula (CZ)	8
		Volyně2	50.445	13.216	B. ×oycoviensis/B. pendula (CZ)	4
					B. ×oycoviensis (CZ)	33
	Křivoklátsko	Bušohrad	49.951	13.813	B. ×oycoviensis/B. pendula (CZ)	1
	Truba Research Station	Truba	50.006	14.836	‘cult. B. szaferi’3	3
Poland		Kobylany2	50.155	19.760	B. atrata	1
					B. obscura	1
					B. ×oycoviensis/B. pendula (PL)	1
					B. ×oycoviensis (PL)	16
					B. pendula (PL)	5
		Kraków (botanical garden4)	50.062	19.959	B. humilis	1
					B. ×oycoviensis/B. pendula (PL)	2
					B. ×oycoviensis (PL)	2
		Skielek2	49.613	20.464	B. ×oycoviensis (PL)	7
					B. pendula (PL)	5
		Kraków (herbarium5)	50.066	19.995	B. szaferi6 (herbarium)	1
TOTAL						106

1Specimens of the Czech origin transplanted from Volyně;

2locality considered as a place of the natural occurrence of B. ×oycoviensis;

3specimens with some traits of B. szaferi cultivated from seeds of the Ojców birches in Volyně;

4The Botanical Garden of the Jagiellonian University;

5Herbarium of W. Szafer Institute of Botany, Polish Academy of Sciences;

6Holotype (KRAM 303846) of B. szaferi.

The sampling of B. humilis was permitted by document no. DZP-WG.6400.6.2020.EP.2 (Generalny dyrektor ochrony Środowiska, Warsaw) and sampling of B. ×oycoviensis was permitted by documents no. OP-I.6400.15.2018.KW (Regionalny dyrektor ochrony Środowiska, Krakow) and no. DZP-WG.6400.23.2018.ep (Generalny dyrektor ochrony Środowiska, Warsaw).

Morphological analyses

Whenever possible, we collected two branches from each sampled individual for analysis of leaf morphology using telescopic shears. From each branch, we randomly selected two leaves for taking measurements (in total, we assessed four leaves per each analyzed individual). We measured following traits on each leaf: blade length [mm], blade width [mm], blade fitting angle [°], blade tip angle [°], leaf serration angle [°], petiole length [mm], distance of the widest part of the blade from the blade base [mm], number of leaf veins [-], distance between the 3rd and 4th vein [mm], number of leaf teeth between the 3rd and 4th vein [-], blade width in the upper 1/4 [mm], distance from the leaf base to the 1st tooth [mm], basal angle [°], 1st vein angle [°], 4th vein angle [°] and distance from the 4th vein to the tip [mm]. We calculated the final values for each individual and each parameter were calculated as averages of four measurements. In the cases of samples of ‘cultivated B. szaferi’ from the Truba Research Station we obtained and averaged only two measurements because it was not possible to take two branches from these samples (the individuals were too young), and in the case of B. szaferi from the herbarium of PAS we obtained only two measurements because we used a scanned image of the herbarium specimen. We analyzed this specimen by taking measurements of its image using the software ImageJ 1.52 [38]. We did not measure the morphological features of the benchmark taxa (B. nana and B. humilis), because it was not possible to measure all parameters on the leaf (4th veins were not present on their leaves). Except for B. szaferi (Herbarium), we measured all specimens using a simple ruler and protractor. We did not include some individuals in the morphological analyses, because it was not possible to take enough material under field conditions; for example, the crown was sometimes too high to allow the sampling of sufficiently developed, insolated leaves for morphometric measurements (e.g. B. atrata and B. obscura in our study). Further methodological information on the measuring of morphological parameters of birch leaves are provided by previous studies [39, 40]. In total, we selected 87 individuals for morphological analyses.

Genome size analysis

The genome size of sample plants was determined by propidium iodide flow cytometry [41] with Solanum pseudocapsicum L. (2C = 2.61 pg) as the internal standard. Leaf tissue from two petioles of each sample was chopped together with about 1.5 cm2 of S. pseudocapsicum leaf tissue in 0.5 ml of Otto I buffer [42]. The resulting suspension was filtered through a 42-μm nylon mesh and left still for ca 20 minutes at 20°C. After that, the suspension was stained with a solution of the following composition: 1 ml of Otto II buffer [42], β-mercaptoethanol (2μl/ml), propidium iodide and RNase IIA.

Flow cytometry was performed using a Partec CyFlow flow cytometer (Partec, Germany) equipped with a green solid-state laser (Cobolt Samba, 532 nm, 100 mW). Holoploid genome size and 1Cx-values (i.e. holoploid genome size divided by the number of chromosome sets [43]) was computed from raw cytometric data using FloMax software and evaluated statistically.

Molecular analyses (SSR genotyping)

Before DNA extraction, individual samples (leaves), stored at −80°C, were frozen in liquid nitrogen and ground by an oscillation mill Retsch MM400 (Retsch GmbH, Haan, Germany). DNA was then extracted from the powdered samples using the QIAGEN DNEasy Plant Mini Kit (QUIAGEN, Hilden, Germany) following the standard protocol. The concentration and quality of DNA was checked prior to PCR using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, Massachusetts, USA) spectrophotometer. For PCR, each DNA sample was diluted to a concentration of 10 ng/μl.

The genetic diversity of the taxa under study was assessed by microsatellite analysis of nuclear DNA. Microsatellite markers were selected based on publications by Tsuda et al. [34, 35] and Kulju et al. [33]. In total, 50 markers were tested, from which 12 polymorphic loci were selected (see S2 Table) and optimized for two multiplex groups, identically as in a previous study by Kuneš et al. [32].

The PCR reactions were run in a total volume of 20 μl: 15 ng of DNA, primers (0.25 μM of each primer), 200 μM of dNTP, 2.5 mM MgCl2 and 1 μM buffer PCR multiplex mix with polymerase.

The PCR program according to Kulju et al. [33] consisted of an initial 5 minutes of denaturation (95°C), which activated the hotStarTaq polymerase, followed by 30 cycles of denaturation (95°C, 60 s), annealing (57°C, 75 s) and elongation (72°C, 150 s). The final extension step took 10 minutes (72°C).

In the PCR program following Tsuda et al. [34, 35], 30 cumulative cycles were performed: denaturation (95°C, 30 s), annealing (55°C, 30 s) and elongation (72°C, 45 s) with a final extension step (7 minutes, 72°C).

The volume of 1 μl of PCR product was added to 14 μl of a solution of formamide with GeneTrace500 DNA ladder (Carolina Biosystems, Prague, Czechia), prepared according to the manufacturer’s protocol. The solution was denatured (5 minutes, 95°C) and quickly cooled on ice.

For determining the length of the amplicons containing the microsatellites, the genetic sequencer Genetic Analyser 3500 (Applied Biosystems, Foster City, California, USA) was used. Raw sequence data were analyzed by GeneMapper 4.1 software (Applied Biosystems, Foster City, California, USA).

Statistical analyses and computations

Morphometric analysis

To compare foliar features between groups (taxa/working units), we performed multivariate analysis of variance (MANOVA) and canonical discriminant analysis (CDA). The results of the CDA are presented as scatterplots. We evaluated the accuracy of CDA discrimination based on ratios of successfully classified individuals for each working group. We performed all computations in R software [44] and produced scatterplots using the R package ‘ggplot2’ [45] and the MorphoTools function set for R [46].

We tested for differences in individual parameters between unequivocally identifiable B. pendula and B. ×oycoviensis individuals across all populations taken together using the t-test (when all assumptions were met) or the Wilcoxon rank-sum test (when they were not). We evaluated the normality of our data was using the Shapiro-Wilk normality test. When the assumption of normality was met but the datasets differed significantly in variances (tested by the F-test of equality of variances), we used Welch test instead. We performed all statistical computations in R [44], with the significance level set to α = 0.05.

Genome size analysis

We did not evaluate the benchmark species (B. nana, B. humilis) and dark-barked birches (B. atrata and B. obscura) in this analysis, as only a few samples of these taxa were available. The Betula szaferi sample obtained from the Kraków herbarium was analyzed without success, as it was impossible to perform flow cytometry on this old dry sample. Flow cytometry was also not successful for five samples of B. pendula from Czechia, one sample of B. ×oycoviensis from Poland, two samples of B. ×oycoviensis from Czechia and one sample of ‘cultivated B. szaferi’. Therefore, flow cytometric analysis was accomplished in 92 samples.

Differences in 1Cx values between working groups were tested for by a Kruskal-Wallis test with multiple comparisons [47], as the assumptions of ANOVA were not met. A box-plot presenting statistically significant differences was produced in R software [44] using the package ‘ggplot2’ [45]. All statistical tests were performed using the significance level of α = 0.05.

Molecular analyses

Genotype data were checked for errors and invalid records. Basic parameters (total allele count, number of used loci and total number of individuals) were evaluated together with basic F-statistics (FST, FIS, FIT) [48] between selected taxa (working groups). For each of the selected taxa (working groups), the numbers of private alleles (those which did not occur in any other group at the respective loci) across all loci were also evaluated.

To visualize relationships between individuals in the study, discriminant analysis of principal components (DAPC) [49] was performed. The structure of the dataset was also evaluated by clustering analysis in STRUCTURE 2.3.4 [50-52] using the following settings: K– 1 to 15 (number of groups + 3), number of runs for each K– 30, burn-in– 100,000 repeats, MCMC repeats– 100,000 repeats. An admixture model with correlated allele frequencies was used. The remaining parameters were left at their default settings. The actual number of populations in the dataset was determined by the method presented by Evanno [53] using Structure Harvester [54]. Cluster matching and permutation was performed in CLUMPP 1.1.2, utilizing the Greedy algorithm with 1,000 repeats [55]. STRUCTURE plots were produced using DISTRUCT [56]. Testing for differentiation between selected taxa (working groups) was done using Goudet’s G-statistic Monte Carlo test [57].

All computations except for those performed in STRUCTURE, CLUMPP and DISTRUCT software were done in R [44], using the packages ‘adegenet’ [58, 59], ‘poppr’ [60, 61] and ‘hierfstat’ [62]. R plots were made using the package ‘ggplot2’ [45].

Results

Morphometric analysis

As is apparent from the CDA scatterplot (Fig 2), there was relatively low variability in morphological traits of leaves among most of the individuals except for the sample of B. szaferi (taken from the herbarium), which is clearly separated on the CDA scatterplot. When the original sample of B. szaferi was excluded from the analysis, samples of ‘cultivated B. szaferi’ appeared outside the point cloud. After the removal of B. szaferi, also the distinction between B. ×oycoviensis and B. pendula, and to some extent between the Czech and Polish populations of B. pendula and B. ×oycoviensis, became more visible. Significant differences between B. pendula and B. ×oycoviensis were identified in several leaf parameters by statistical analyses (Table 2).

Fig 2

Results of canonical discriminant analysis (CDA) of morphological traits of leaves presented as a scatterplot of selected birch individuals.

Plot A depicts the results of CDA analysis of all samples included and plot B depicts the results of CDA analysis without the holotype of B. szaferi, which appeared as an outlier in the first CDA analysis. The percentages in axis titles stand for percentages of explained variation by the respective axis.

Table 2

Results of testing for differences between B. pendula and B. ×oycoviensis in individual foliar parameters.

Parameter	Mean (B. oyc.)	Mean (B. pen.)	Test	Test stat. value	p-value	Sig.
Blade length [mm]	33.9	43.1	N	239.5	< 0.001	***
Blade width [mm]	25.6	33.9	N	235.0	< 0.001	***
Blade fitting angle [°]	292.6	299.6	N	553.5	0.115	ns.
Blade tip angle [°]	47.5	40.1	P*	3.485	<0.001	***
Leaf serration angle [°]	74.6	63.2	P*	3.751	< 0.001	***
Petiole length [mm]	15.2	15.8	P	0.823	0.413	ns.
Distance of widest part of blade from blade base [mm]	11.6	13.7	N	440.0	0.006	**
Number of leaf veins [–]	6.0	7.8	N	204.0	<0.001	***
Distance between 3rd and 4th vein [mm]	4.8	6.0	N	294.0	<0.001	***
Number of leaf teeth between 3rd and 4th vein [–]	1.6	2.7	N	289.0	<0.001	***
Blade width in the upper 1/4 [mm]	10.2	12.0	N	403.0	0.002	**
Distance from the leaf base to the 1st tooth [mm]	11.7	14.3	N	291.0	< 0.001	***
Basal angle [°]	245.3	239.2	N	889.5	0.055	ns.
1st vein angle	56.4	65.1	P*	3.532	<0.001	***
4th vein angle	30.3	32.5	P	2.109	0.038	*
Distance from the 4th vein to the tip [mm]	19.1	31.0	N	197.5	<0.001	***

Notes: N in the column ‘Test’ stands for the non-parametric Wilcoxon rank-sum test, P stands for the parametric t-test. P* stands for the Welch t-test. Significance level symbols: ns.–not significant,

*–p < 0.05,

**–p < 0.01,

***–p < 0.001.

Only clearly distinguishable individuals were analyzed. Samples from Czechia and Poland were analyzed together. In total, 47 samples of B. ×oycoviensis and 30 samples of B. pendula were analyzed.

The testing for differences in leaf parameters between selected working groups yielded significant results (MANOVA, df = 7, Pillai’s trace = 2.902, p < 0.001). The accuracy of CDA discrimination was 100% except for B. ×oycoviensis from the Czechia (74.2%), B. ×oycoviensis from Poland (81.3%) and B. pendula from Poland (70.0%).

Trees classified as B. pendula and B. ×oycoviensis (those which showed at least 80% of traits described in the relevant literature, see the Material and methods) differed significantly (p < 0.05) from each other in 13 out of the 16 morphometric parameters analyzed (see Table 2).

Individuals of B. pendula from Czechia (the Ore Mts. and surroundings) possessed greater 1Cx values (median: 0.4413) compared to the other groups analyzed, including Polish B. pendula (median for the other groups together: 0.4280). The Kruskal–Wallis test revealed significant differences (chi-squared = 32.313, df = 6, p < 0.001) in 1Cx values. The results of multiple comparisons are depicted in Fig 3.

Fig 3

1Cx values of samples of selected taxa/working units with results of multiple comparisons.

Groups (taxa / working units in the Poland and Czechia) denoted by different letters exhibited significant differences in 1Cx values at α = 0.05. The box and whiskers plots are presented in standard Tukey’s design: Whiskers depict the minimum and maximum excluding outliers, black dots represent outliers (less than the lower quartile—1.5 times the inter-quartile range and more than the upper quartile + 1.5 times the inter-quartile range, respectively).

Molecular analyses

Altogether, 116 alleles at twelve loci were identified in a total of 106 individuals. F-statistics indicated very low overall variability (FIT = 0.1088), 23% of which accounted for among-population variability (FST = 0.0254, FIS = 0.0834). After the removal of the two benchmark taxa (B. nana and B. humilis) from the dataset (see below for more details), FST accounted for only 12% of the variability, suggesting that all the other groups form quite a homogeneous complex (FST = 0.0105, FIS = 0.0799).

Private alleles were found in seven groups out of the twelve groups examined. The greatest numbers of private alleles per individual were found in the cases of B. humilis and B. nana (benchmark taxa). Among the other groups, the greatest number (proportion) of private alleles per individual was found for B. pendula from Czechia (Table 3).

Table 3

Private alleles of selected groups across all twelve loci.

Group (taxon/working unit)	Private alleles	Individuals	Private alleles per individual
B. ×oycoviensis (CZ)	6	35	0.17
B. ×oycoviensis (PL)	2	25	0.08
B. pendula (CZ)	10	20	0.50
B. pendula (PL)	1	10	0.10
B. humilis	5	1	5.00
‘cult. B. szaferi’	2	3	0.67
B. nana	4	1	4.00

The overall DAPC analysis (with all samples included in the analysis) clearly separated the benchmark taxa (B. nana and B. humilis) whereas all other groups formed a compact cloud with relatively low variation (Fig 4).

Fig 4

Scatterplot from DAPC analysis of all individuals. Benchmark taxa, which were the only taxa to be clearly distinguished by the DAPC, are labelled in the plot.

When the two outlier points representing benchmark taxa were removed from the analysis, no clear pattern between the other taxa was detected (Fig 5).

Fig 5

Scatterplot of individuals excluding benchmark taxa (B. nana and B. humilis) produced by the DAPC method.

Differences between the groups in the dataset without the benchmark taxa were statistically tested for by G-statistics, which returned non-significant result (100,000 iterations, p = 0.68).

The homogeneity between all groups (working units) except for B. nana and B. humilis was also supported by analyses in STRUCTURE and CLUMPP software. The number of clusters in the whole dataset was set to 4, based on an analysis according to Evanno et al. [53] in Structure Harvester [54]; for more information, see S2 Table. The benchmark taxa were included in a separate group (denoted by orange color in Fig 6) whereas all other groups formed quite a homogeneous cluster.

Fig 6

Results of analyses in STRUCTURE and CLUMPP software.

Each bar depicts one individual and its estimated probability of affiliation to a group denoted by its color. The number of groups in the dataset was estimated to be 4 using the method of Evanno et al. [53] using STRUCTURE HARVESTER [54]. The plot was created by DISTRUCT software [56].

Discussion

The Ojców birch (B. ×oycoviensis Besser) is one of the birch taxa whose taxonomic treatment remains unresolved. It was first described by Besser [12], and studies carried out in the 20th century [15-17] suggested that B. ×oycoviensis is a hybrid species between B. pendula and ‘B. nova’ [15]. The latter putative parent was later validly described as Betula szaferi by Jentys-Szaferowa ex Staszkiewicz. By giving the epithet ‘szaferi’ to ‘B. nova’, Jentys-Szaferowa wished to commemorate her husband, Polish botanist Władisław Szafer [19].

Like Staszkiewicz [19], we found, at sites of reported occurrences of B. ×oycoviensis the individuals showing the traits of B. ×oycoviensis scattered across stands of typical B. pendula or mixtures of typical B. pendula and other tree genera (e.g. Picea, Acer and Fagus). Our search for B. szaferi in the wild of Czechia and Poland was not successful.

However, we examined several individuals resembling B. szaferi that were grown from seeds of specimens classified as B. ×oycoviensis, sampled in Czechia (locality Volyně u Výsluní). Similar results were obtained by Jentys-Szaferowa [28], who described three types of progeny obtained by controlled crossings of B. ×oycoviensis: type oycoviensis, type pendula and type ‘nova’ (lately ‘szaferi’). Both B. ×oycoviensis and B. szaferi were originally classified as species. The existence of three types of progeny could imply a ‘hybrid’ origin of B. ×oycoviensis but is definitely not sufficient to prove such a hypothesis. Moreover, no difference between B. szaferi and B. pendula have been observed using molecular methods (see Figs 4–6). Therefore, B. ×oycoviensis could have originated by the crossing of visually somewhat distinct parents belonging to the population of B. pendula. This theory would also explain the occurrence of B. ×oycoviensis in countries other than Poland. The question is what traits actually differentiate species from taxa of lower taxonomical ranks, which is rather a conceptual issue [63, 64], and how these traits are controlled and linked.

The testing for differences in leaf morphology via MANOVA yielded significant results. Subsequent CDA analysis revealed distinct separation of the sample of B. szaferi taken from the Krakow herbarium. The separation of this particular sample is not surprising, as the peculiarity of its leaf shape is apparent to the naked eye. When the sample of B. szaferi was removed from the CDA analysis, samples of ‘cultivated B. szaferi’ appeared as outliers, which could be expected too, as those samples were selected based on their distinctive leaf morphology. The overall leaf shape of the samples of B. pendula and B. ×oycoviensis (including samples with mixed traits) showed some minor differences; for example the Czech population of B. ×oycoviensis as that of B. pendula appeared to be slightly distinctive from its Polish counterparts. This could be a ‘random effect’ of geographical distance, but it could also be a result of minor phenotypic and genetic differences, possibly reflected in slightly different genome size of the Czech population of B. pendula compared to that of other selected working units (see below). This topic, however, surely merits a detailed study, and our data are not sufficient to draw any definite conclusion.

Our testing of individual morphological traits revealed significant differences between clearly identifiable samples of B. pendula and B. ×oycoviensis in thirteen parameters out of the sixteen tested, namely all parameters tested except for blade fitting angle [°], petiole length [mm] and basal angle [°]. These outcomes of our morphological studies are more or less consistent with the results obtained by Baláš et al. [27], who found significant differences in seven out of sixteen morphological traits, but it is important to mention that the study by Baláš et al. [27] was performed only on samples collected at Volyně u Výsluní, Czechia. Blade length and the number of leaf veins are also mentioned as important for the determination of B. ×oycoviensis by the keys to the floras of Poland [20] and Czechia [65], together with the growth of leaves in groups on brachyblasts in B. ×oycoviensis. An overview of the whole habitus is needed for the determination of B. ×oycoviensis in the field, as the leaf shape differences between the taxa in question do not have to be distinctive enough. Some habitus traits are also described in the keys to the floras mentioned above. Still, many individuals exhibit mixed traits of B. pendula and B. ×oycoviensis, so it is quite difficult to classify them [66]. That is also the main reason why we distinguished the B. pendula/B. ×oycoviensis working unit in this study.

Our genome size analysis revealed no significant differences between the defined groups except for B. pendula originating from the Czech population (the Ore Mts. and their surroundings), which displayed significantly greater values compared to the other groups. Because B. pendula from Czechia does not differ from other groups in the results of our molecular analyses, the reason behind the differences in genome size could reside not only in intraspecific ploidy-related or monoploid-level variation [67] but also in some kind of chromosomal disorder. We, however, attribute these differences to intraspecific variability because our as yet unpublished data indicate relatively extensive variation in genome size (1Cx values, respectively) between populations of B. pendula in Czechia (see Fig 7). However, the difference could easily be also a result of some level of methodological inaccuracy. Therefore, a survey focused on genome size across B. pendula populations may be desirable to check for the existence of a pattern of genome size with respect to geographic distribution.

Fig 7

Comparison of genome size of selected populations of B. pendula from Czechia, including some hitherto unpublished data obtained by an identical procedure as described in the material and methods section.

Relatively close locations within Central Bohemia were selected to illustrate the relatively large genome size variability on a small area; samples from Southern Moravia are included as an outlier population. The number of samples analyzed is indicated below each box. The box and whiskers plots are presented in standard Tukey’s design: Whiskers depict the minimum and maximum excluding outliers, black dots represent outliers (less than lower quartile—1.5 times the inter-quartile range and more than upper quartile + 1.5 times the inter-quartile range, respectively).

Our molecular analyses detected relatively low inter-group variability, which suggests that all of the groups considered are genetically highly homogeneous (including the Polish and Czech B. ×oycoviensis and B. pendula). The same results concerning B. ×oycoviensis were reported in a previous paper by Kuneš et al. [32], based, however, only on a small number of B. ×oycoviensis samples from Czechia. The only clearly distinguished samples were those of the diploid benchmark species (B. humilis and B. nana), which were visibly separated from the other samples in the DAPC scatterplot (Fig 4). When these benchmark taxa were removed, only small differences could be observed, for example between samples of ‘cultivated B. szaferi’ and samples of B. ×oycoviensis from Poland or between samples of B. ×oycoviensis from Czechia and Poland. However, in spite of some gentle difference, the Polish and Czech populations of the Ojców birch most probably belong to the same taxon. The low-level variation is probably caused by some population variability or random effects.

Aside from the benchmark taxa, the greatest mean number (proportion) of private alleles per sample was found in case of B. pendula sampled in Czechia (0.5), which is in line with the observed difference in genome size. This fact might suggest that B. ×oycoviensis originated from B. pendula which was ‘slightly different’ from B. pendula sensu stricto, but as already said, further research is needed to draw solid conclusions. Nevertheless, it can be stated that B. pendula, B. ×oycoviensis and B. szaferi in our dataset do not differ genetically at the species level. Ashburner & McAllister [2] argued that the Ojców birch is a weak growing, precocious and heavily fruiting form of B. pendula. More recently, the chapter on birches in the Flora of the Czech Republic [21] classified B. ×oycoviensis as a variety of B. pendula–B. pendula var. oycoviensis. The experimental data summarized in our study support these opinions.

In Poland, B. ×oycoviensis is a species strictly protected by law (Ministry Decree: Poz. 1409 Rozporządzenie ministra środowiska z dnia z dnia 9 października 2014 r. w sprawie ochrony gatunkowej roślin). In Czechia, this taxon is included on the Red List of Vascular Plants, although it is not protected by law [68]. On the other hand, the Ojców birch is included neither on the European Red List of Trees [69] nor on the Red List of Betulaceae [70]. The exclusion of the Ojców birch from the latter two Red Lists might be attributable to its expected downgrading from the species level to a significantly lower taxonomic rank.

Nonetheless, we are convinced that the Ojców birch still deserves some level of protection and distinction in terms of conservation regardless of the taxonomic rank at which this birch is most appropriately classified. It is an interesting birch with a characteristic ‘broomy’ habitus whose origin is yet to be completely resolved. To answer the remaining questions or to test standing hypotheses related to the Ojców birch and its putative parent (B. szaferi), we should keep protecting this rare taxon and its habitats. At present, conservationists are reducing the emphasis on species conservation and are becoming increasingly aware of biodiversity at all levels of the hierarchy of life [64]. We suggest that this should be reflected on some reasonable level also in case of the Ojców birch, at least before its taxonomic position is resolved satisfactorily. In our opinion, this holds true, especially if the rare populations of the Ojców birch in Czechia and Poland diminish in size. A good example of the usefulness of this approach is that of the curly birch (Betula pendula Roth. var. carelica [Merklin] Hämet-Ahti), whose unique traits are highly valued even though its taxonomic value may be low.

Conclusions

Betula pendula and B. ×oycoviensis in our dataset do not differ genetically at the species level despite being distinct morphologically. On a scatterplot produced by the DAPC method, excluding diploid benchmark species (B. nana and B. humilis), the holotype of B. szaferi rests in a cloud consisting of individuals of B. pendula and B. ×oycoviensis, although it is shifted to the part of the cloud represented by specimens of Czech origin.

Our molecular analyses detected low variability between the groups under comparison (taxa and working units of the Czech and Polish provenance) after the exclusion of the benchmark species. This low variability suggests that the Polish and Czech populations of B. ×oycoviensis are genetically very close even though some small differences related to geographic origin may be traced. The classification of the Ojców birch as B. pendula var. oycoviensis seems more accurate than its treatment as a separate hybridogenous species under the name B. ×oycoviensis.

Holotypus of B. szaferi sampled in herbarium of the W. Szafer Institute of Botany, Polish Academy of Science (PAS) in Kraków (KRAM 303846).

(JPG)

Click here for additional data file.

Basic description of all taxa involved in the study.

(DOCX)

Click here for additional data file.

Estimation of K (the number of ‘groups’ in microsatellite data) using the method of Evanno et al. (2005).

(XLSX)

Click here for additional data file.

The description of used microsatellite markers.

(XLSX)

Click here for additional data file.

(XLSX)

Click here for additional data file.

18 Sep 2020

PONE-D-20-26980

Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: a revised view of the taxonomic treatment of the Ojców birch

PLOS ONE

Dear Dr. Linda,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The main shortage of the manuscript is that the authors used an unsubstantial sample set of the studied taxon. The Material and Methods section lacks clear elaboration of the methods used, in the first place statistical assessment. Inadequately analyzed and presented results may hide many valuable conclusions. It is also recommended to perform additional analysis of the morphometric data (Reviewer #1).

Both Introduction and Discussion sections must be thoroughly revised according to the reviewers' reports. Headings and subheadings should be defined according to the place in the manuscript and the content of the chapter (e.g. three subheadings are stated as: "Morphological analyzes").

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Reviewer #1: Partly

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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Reviewer #1: The manuscript deals with the endemic taxon of the genus Betula occurring primarily in southern Poland but also in the Bohemian Massif of the Czech Republic. Positive feature of the manuscript is that the authors used several techniques (morphometrics of leaves, flow cytometry and molecular analyses) to describe the taxonomic position of Betula oycoviensis.

As I understood correctly, the Ojców birch is considered to be a putative hybrid species between Betula pendula and B. szaferi and as such it is an endemite for southern Poland. I wonder, how is it possible that this species also occurs in remote parts of Europe in Denmark and Sweden and/or Romania and Ukraine. If the hybrid origin should be true, then in all the above mentioned countries both putative parent species should be present (or they were present in the past). The other option could be that the origin of Ojców birch fits completely into the variation of Betula pendula.

The authors used rather limited sample sizes both for Betula oycoviensis, and for the species they have compared it with, e.g. Betula pendula. I understand that the natural populations of Betula oycoviensis are not very numerous, but on the other hand, they could have done more attempts to produce reliable number of progenies in order to find putative parents. Progeny test based on three individuals is not very trustworthy. The same is true for the individuals growing in the botanical gardens. If you interpret the data based on this experiment, you should be very careful.

As for the morphometrics of the leaves, the information whether the leaves have been chosen from brachyblasts or macroblasts in order to make proper comparison with individuals from the progeny tests is missing. There should not be a problem to get reliable experimental material from high trees (lines 151–153). There are still possibilities to shoot down branches, if there is a problem with climbing the trees. As for the morphometric analyses of leaves, the authors measured a set of linear and angular traits and numbers, thus producing a data set suitable for multidimensional data analyses. However, only PCA is not enough for the morphometric analysis. I would recomend to study the approach published in Koutecký (2015), where the morphometric data were analysed using "MorphoTools" R scripts in R software. Hence, Canonical Discriminant Analyses (CDA) would be employed to select morphological characters which preferably separate ahead selected groups. Also, the nonparametric k-nearest neighbour approach could be used for classificatory discriminant analysis. See: Koutecký P. (2015): MorphoTools: a set of R functions for morphometric analysis. Plant Systematics and Evolution 301: 1115–1121.

More details about the genome size estimation should be given. How many replicates of each samples were carried out? Did the genome size estimation followed “best practice” three replicates on different days as suggested by Doležel et al. (2007) Nat Protoc? The information on the concentrations of mercaptoethanol for Otto II (lines 161–164) is missing. There is no information where the chromosome numbers by which you divided the holoploid (1C) in order to get Cx (monoploid) genome were taken from. Did you count chromosomes? Do you have any plant with known chromosome number to which others measured by flow cytometry were standardized?

I wonder why the author did not use (or at least test) some of the uniparentaly inherited molecular markers (cpDNA) in order to look properly at the origin of putative parents or the way of putative hybridization history.

Major improvement of this manuscript is needed in statistical processing of multidimensional morphometric data e.g. using the discriminant analysis.

Notes

Ln 18 Ojców

Ln 35–37 refrase the sentence

Ln 64 insert comma prior “Czerwona Góra”, i.e. “, Czerwona Góra”

Ln 112 replace “the herbarium of the Polish Academy of Sciences in Kraków” by “herbarium in Kraków” or “Kraków herbarium”. The complete name of this herbarium is already given in lines 100–101

Ln 140–141, 148–149 parts of both sentences are repeated

Ln 162, 171, 184–193 insert space after number (e.g. 20 °C)

Ln 170 Molecular analyses (SSR genotyping)

Ln 172, 174 and others Retsch MM400 should follow the producing company, city and country e.g. DNAeasy Blood and Tissue kit (Qiagen, Hombrechtikon, Switzerland).

Ln 177 microsatellite analysis of nuclear DNA

Ln 199 and 249 Morphometric analysis

Ln 213–214 Information in the first sentence is already given earlier, ln 166–168.

Ln 372, 378 characters or traits instead of parameters

Ln 371–376 You write about the results of univariate tests. What about using the multivariate analysis, e.g. canonical discriminant analysis with subsequent classification of individuals into groups (species and/or population and species).

Ln 473 B. szaferi

Ln 490–492 remove capital letters. Morphological variation among Betula nana (diploid), B. pubescens (tetraploid) and their triploid hybrids in Iceland.

Ln 572 remove “Nonparametric statistics for the behavioral sciences, 2nd ed.”

Ln 573 replace “Mcgraw-Hill Book Company” by “McGraw-Hill Book Company”

Ln 574 remove capital letters. Genetical structure of populations

References

- Complete “doi” where missing, e.g. Järvinen P, et al., 2004; Ln 484 and some other papers.

- Insert a space after year and semicolon

- Insert a space after the volume (or remove it), it should be unified

- Latin names should be in Italics also in References (although this rule is not followed in all papers published in PLoS ONE).

Reviewer #2: Section Introduction:

I have found that the entire "introduction" section is hard to understand clearly. It is not unequivocally clear which taxa are in question. Also, the literature about the locations of taxa of interest is not clearly stated. The introduction section does not clearly describes the problem related to taxonomic status of the Oycow birtch.

Also, in the text of introduction, there is nothing stated about the morphometric and genetic analyses that are used in the research; or the approaches used in research either.

The entire section of the introduction must be revised and supplemented with relevant topics covered in the manuscript.

Subsection Sampling:

It is required to change subsection name into “Plant material”.

Line 85: Is the natural origin of the samples obtained from the botanical garden and zoo known?

Line 108: … although the taxonomic significance of these dark-barked birches remains questioned… This statement does not belong to the section M&M. Move to discussion.

Line 117-119: I suggest to the author consider using the term “population”. Terms “working units” and “group” is confusing. If the author still wants to use these terms, he should better explain and define them and not just refer to another reference (line 105-106).

Subsection Morphological analyses:

Line: 131: …. two branches from each sampled individual were collected for analysis …

the sentence does not agree with the statement in the line 115-116.

Subsection Genome size analysis:

line: 158: This method measures the amount of DNA per cell ( in pg in this case) by comparing the amount of DNA to a known internal standard (in this case Solanum pseudocasicum). Genome size refers to the amount of DNA contained in a haploid genome expressed either in terms of the number of base pairs, kilobases (1 kb = 1000 bp), or megabases (1 Mb = 1 000 000 bp), or as the mass of DNA in picograms (1 pg = 10−12 g)( https://www.sciencedirect.com/topics/immunology-and-microbiology/genome-size ). Please reformulate these sentences with the correct use of the term. This applies to the rest of the text. In general: literature data on the level of ploidy of taxa of interest are mentioned just in the line 214. This information should be stated and commented on in a more transparent way, considering that polyploidization the genus Betula is mentioned.

Subsection Molecular analyses

Line 179: Which 12 microsatellite markers did you use? Are they listed somewhere? You used the microsatellite markers listed in the literature. However, there must be an exact list of which markers you used.

Line 194: You did not determine the "microsatellite length". You determined the length of the amplicons containing the microsatellites. The correct use of the terms is an imperative of scientific work.

Section: Statistical analyses and computations/ subsection Morphological analyses

You did not state how the PCA analysis was performed. How did you use the data given in different units of measurement (mm and degrees of angles) in this analysis? Was the data normalized before PCA analysis? How are they normalized?

How did you analyze continuous and discrete data (eg leaf length and nerve number) together in same PCA analysis?

**********

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Reviewer #1: No

Reviewer #2: No

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16 Nov 2020

Manuscript ID: PONE-D-20-26980

Manuscript Title: Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: a revised view of the taxonomic treatment of the Ojców birch

Dear Editor,

We are grateful to the reviewers for their valuable comments. We appreciate the reviewers’ inputs to the manuscript and the time they spent refereeing it. Their inputs have helped to improve the resubmitted document. We edited the document according to instructions in the review comments to meet the requirements of the reviewers and the publication criteria. The updated version of the manuscript was also completely checked by native speaker specialist in the field (see Acknowledgements).

In the following part, we provide point-by-point responses to the reviewer comments (in italics).

Authors

Reviewer #1

General comment

The manuscript deals with the endemic taxon of the genus Betula occurring primarily in southern Poland but also in the Bohemian Massif of the Czech Republic. Positive feature of the manuscript is that the authors used several techniques (morphometrics of leaves, flow cytometry and molecular analyses) to describe the taxonomic position of Betula oycoviensis.

As I understood correctly, the Ojców birch is considered to be a putative hybrid species between Betula pendula and B. szaferi and as such it is an endemite for southern Poland. I wonder, how is it possible that this species also occurs in remote parts of Europe in Denmark and Sweden and/or Romania and Ukraine. If the hybrid origin should be true, then in all the above-mentioned countries both putative parent species should be present (or they were present in the past). The other option could be that the origin of Ojców birch fits completely into the variation of Betula pendula.

The authors used rather limited sample sizes both for Betula oycoviensis, and for the species they have compared it with, e.g. Betula pendula. I understand that the natural populations of Betula oycoviensis are not very numerous, but on the other hand, they could have done more attempts to produce reliable number of progenies in order to find putative parents. Progeny test based on three individuals is not very trustworthy. The same is true for the individuals growing in the botanical gardens. If you interpret the data based on this experiment, you should be very careful.

As for the morphometrics of the leaves, the information whether the leaves have been chosen from brachyblasts or macroblasts in order to make proper comparison with individuals from the progeny tests is missing. There should not be a problem to get reliable experimental material from high trees (lines 151–153). There are still possibilities to shoot down branches, if there is a problem with climbing the trees. As for the morphometric analyses of leaves, the authors measured a set of linear and angular traits and numbers, thus producing a data set suitable for multidimensional data analyses. However, only PCA is not enough for the morphometric analysis. I would recomend to study the approach published in Koutecký (2015), where the morphometric data were analysed using "MorphoTools" R scripts in R software. Hence, Canonical Discriminant Analyses (CDA) would be employed to select morphological characters which preferably separate ahead selected groups. Also, the nonparametric k-nearest neighbour approach could be used for classificatory discriminant analysis. See: Koutecký P. (2015): MorphoTools: a set of R functions for morphometric analysis. Plant Systematics and Evolution 301: 1115–1121.

More details about the genome size estimation should be given. How many replicates of each samples were carried out? Did the genome size estimation followed “best practice” three replicates on different days as suggested by Doležel et al. (2007) Nat Protoc? The information on the concentrations of mercaptoethanol for Otto II (lines 161–164) is missing. There is no information where the chromosome numbers by which you divided the holoploid (1C) in order to get Cx (monoploid) genome were taken from. Did you count chromosomes? Do you have any plant with known chromosome number to which others measured by flow cytometry were standardized?

I wonder why the author did not use (or at least test) some of the uniparentaly inherited molecular markers (cpDNA) in order to look properly at the origin of putative parents or the way of putative hybridization history.

Major improvement of this manuscript is needed in statistical processing of multidimensional morphometric data e.g. using the discriminant analysis.

Authors’ answers to general comment

The primary idea that brought us to this topic was the unclarified taxonomic position of Ojców birch that was treated differently by various literature sources. We have used classical morphometric measurements, SSRs genotyping and Genome size analysis. Our aim was to describe the variability of (in particular) the Czech and Polish populations of Ojców birch and compare these two populations together with samples of silver birch. This research has some practical implications for conservation (see the further text). Therefore, we added some short text to the manuscript explaining this.

The topic has two levels, and we intend to address them stepwise (in more studies) although, we admit, these levels are interlinked: The first level is related to the general taxonomical position of Ojców birch. Shortly: Could Ojców birch considered a species, or (much more probably) should this taxon be classified at lower taxonomic rank? Is the Czech population of Ojców birch the same as the population of Ojcow birch in Poland?

The second level may go more in-depth and focus on the hybridization theory and look properly on putative parents etc. Some malformations caused by external factors could theoretically play a role as well, but this could be a topic for another study.

This assessed manuscript aims at the first level and has some practical implications for conservation management of the taxon in the Czech Republic. There is only one locality in Czechia with the acknowledged population of Ojców birch counting several tens of individuals. Some management steps should be taken for its conservation because the Czech population of Ojców birch grows old, and its natural regeneration is poor on the site. However, these steps should reflect the taxonomical position of Ojców birch. To assess this position on level important for nature conservation, the methods used in our study are, in our opinion, sufficient. We suggest that Ojcow birch could not be a species, and this is documented using diploid benchmark taxa in our study (Fig. 5). The applied methods should be sufficient also for the comparison of the Czech and Polish population. Theoretically, Polish individuals could strengthen the Czech population of Ojców birch. For comparison, we have carefully chosen two important locations in Poland, where Ojców birch was reported: Skielek and Kobylany. In Chojnik only four individuals were reported in 1967, Hammernia is a very small nature area (reserve) close to Kobylany, and Czerwena Gora belongs to another district of Poland and sampling there would require a different administrative process to receive a permit for sampling (Ojcow birch is strictly protected in Poland). As for individuals from the botanical garden, we sampled only individuals that manifested the traits of Ojców birch. Lots of trees grown in the botanical gardens are grown from seed, and many of them do not retain the traits of Ojców birch.

Answer to specific parts of general comment:

…As I understood correctly, the Ojców birch is considered to be a putative hybrid species between Betula pendula and B. szaferi and as such it is an endemite for southern Poland. I wonder, how is it possible that this species also occurs in remote parts of Europe in Denmark and Sweden and/or Romania and Ukraine. If the hybrid origin should be true, then in all the above-mentioned countries both putative parent species should be present (or they were present in the past)...

… Progeny test based on three individuals is not very trustworthy. The same is true for the individuals growing in the botanical gardens. If you interpret the data based on this experiment, you should be very careful.

Betula szaferi is no longer viewed as endemic in Poland (according to Staszkiewicz, 2013); it is, however, considered a parental species of Ojców birch that is allegedly hybridogenous species. Both taxa, B. szaferi and B.×oycoviensis were first described in Poland. Unfortunately, B. szaferi is recently reported as missing in the wild. Even in the Botanical garden in Kraków, there is no longer any living specimen of this taxon, and we did not find any specimen during our detailed survey in Skielek in 2019. Therefore, we used the herbary item of B. szaferi from Kraków (Polish origin) and included the samples of “bred B. szaferi” received from the seed (Czech origin from Volyně) in our research station. We should mention in this regard, that in Kraków, we received a fragment of holotypus (KRAM 303846) for our analyses, see Staszkiewicz (1986) and figure documentation to this answer. The molecular analyses of this holotypus were successful and included in our study. Therefore, we think that our material (despite being limited) provides under the existing circumstances a reasonable base for our manuscript.

In our study, we do not present outcomes of any special progeny test. We included three specimens up to now grown from seeds of the Czech B. oycoviensis in our research station that possess the traits of B. szaferi, next to this we refer to the progeny tests conducted by Jentys-Szaferowa (1967).

To clarify this and avoid misunderstanding, we changed the term “bred B. szaferi” to “cult. B. szaferi” (as “cultivated”).

We are aware of the limitations and mention these in our manuscript: Ln: 111–114 and 363–368 in the first-round version.

…The other option could be that the origin of Ojców birch fits completely into the variation of Betula pendula…

Yes. Our results suggest this view (e.g. Fig. 5), and we support this opinion in our manuscript: Ln: 424–431 of the original version and the closing sentence of the abstract.

… As for the morphometrics of the leaves, the information whether the leaves have been chosen from brachyblasts or macroblasts in order to make proper comparison with individuals from the progeny tests is missing.

We avoided sampling apical leaves or leaves situated close to the apex of shoots or catkins, we tried to sample well-developed insolated leaves. However, we did not distinguish the position on brachyblast or macroblasts. In the case of B. pendula, to keep sampling from brachyblasts would be difficult, since these are much less frequent (often missing) on the shoots of. B. pendula than in other taxa, at least in case of our samples of. B. pendula. On the other hand, in the case of B. oycoviensis, most of the characteristic small leaves are placed on the brachyblasts. Neither we have recorded any recommendation to distinguish brachyblast or macroblast leaves in the studies focused on distinguishing of birches using the traits of foliar morphology. The word “randomly” was added to the sentence at the beginning of the section “Morphological analyses” to show that we did not distinguish brachyblast vs. macroblast leaves.

There should not be a problem to get reliable experimental material from high trees (lines 151–153). There are still possibilities to shoot down branches, if there is a problem with climbing the trees

Technically, it is the truth. However, according to Polish law, we sampled strictly protected species. To receive permission (which is quite a long-term process), we had to sample a precisely defined amount of material (length of shoots) from each tree, and the technique of sampling had to be described in advance already in the application. We did not dare to mention shooting down the shoots in the application. For tree sampling, we used telescopic shears, which were ca 5 m long, but unfortunately, some trees had insolated part of crowns situated much higher, and we were not able to take samples that were enough representative for foliar morphology. The number of silver birch samples are limited because we have tried to take samples from populations in the close neighborhood of the localities of the Ojców birch, however, at the same time far enough from such localities so that the hybridization could be excluded (or at least were extremely improbable). When implementing the revisions, to make our outcomes in morphology more precise, we measured once more the older samples, so that all the samples were measured by the same person (e.g. Tab. 2, Fig. 2). Relevant statistics were recalculated using updated data.

I wonder why the author did not use (or at least test) some of the uniparentaly inherited molecular markers (cpDNA) in order to look properly at the origin of putative parents or the way of putative hybridization history.

We have used microsatellites for the analysis, as this is widely used approach and also, we already have optimized method (e.g. Kuneš et al., 2019, https://doi.org/10.1371/journal.pone.0224387).

As for the morphometric analyses of leaves, the authors measured a set of linear and angular traits and numbers, thus producing a data set suitable for multidimensional data analyses. However, only PCA is not enough for the morphometric analysis. I would recomend to study the approach published in Koutecký (2015), where the morphometric data were analysed using "MorphoTools" R scripts in R software. Hence, Canonical Discriminant Analyses (CDA) would be employed to select morphological characters which preferably separate ahead selected groups. Also, the nonparametric k-nearest neighbour approach could be used for classificatory discriminant analysis. See: Koutecký P. (2015): MorphoTools: a set of R functions for morphometric analysis. Plant Systematics and Evolution 301: 1115–1121.

For the morphometric analyses, we have used some parts of MorphoTools script, thank you for the information about this tool. We have provided CDA analysis (partly from the MorphoTools script) as a substitute to previously presented PCA analysis.

…More details about the genome size estimation should be given. How many replicates of each samples were carried out? Did the genome size estimation followed “best practice” three replicates on different days as suggested by Doležel et al. (2007) Nat Protoc? The information on the concentrations of mercaptoethanol for Otto II (lines 161–164) is missing. There is no information where the chromosome numbers by which you divided the holoploid (1C) in order to get Cx (monoploid) genome were taken from. Did you count chromosomes? Do you have any plant with known chromosome number to which others measured by flow cytometry were standardized?

We have done one flow cytometry run per sample. We did not conduct three replicates on different days. There were technical reasons to make only one flow cytometry run per sample: The analyses were not conducted in our facilities but in the laboratory of the Charles University in Prague (see Acknowledgements) to keep the same laboratory and equipment for all analyses in this study. Consequently, there were not as many term opportunities in this laboratory of the Charles University to conduct three replications. Moreover, we analyzed fresh material whose storage time was limited. The sampling and transport of the fresh samples from Poland consumed some significant time. Thus the remaining time available prior to deterioration of samples’ quality did not enable us to conduct analyses on more days. If some doubts about the reliability of the outcomes appeared during processing in the laboratory, the analysis was repeated immediately.

The concentration of mercaptoethanol was added into the text. We did not count chromosomes, we used Solanum pseudocapsicum as an internal standard, as it is stated at the beginning of the “Genome size analysis” subsection of our manuscript. Some of older samples of B. pendula were resampled and analyzed once more using Solanum pseudocapsicum as an internal standard at the beginning of 2020 so that the standard was the same for all samples included in the study.

Specific comments and answers

Ln 18 Ojców - Corrected

Ln 35–37 refrase the sentence – The sentence was rephrased

Ln 64 insert comma prior “Czerwona Góra”, i.e. “, Czerwona Góra” – Comma inserted

Ln 112 replace “the herbarium of the Polish Academy of Sciences in Kraków” by “herbarium in Kraków” or “Kraków herbarium”. The complete name of this herbarium is already given in lines 100–101 – Changed for “the herbarium in Kraków”

Ln 140–141, 148–149 parts of both sentences are repeated – Thank you for your comment, the second (repeating) sentence was deleted from the manuscript.

Ln 162, 171, 184–193 insert space after number (e.g. 20 °C) – the space was inserted in all occurrences

Ln 170 Molecular analyses (SSR genotyping) - corrected

Ln 172, 174 and others Retsch MM400 should follow the producing company, city and country e.g. DNAeasy Blood and Tissue kit (Qiagen, Hombrechtikon, Switzerland). – address information added to all described products

Ln 177 microsatellite analysis of nuclear DNA – information added

Ln 199 and 249 Morphometric analysis - corrected

Ln 213–214 Information in the first sentence is already given earlier, ln 166–168. – Sentence deleted

Ln 372, 378 characters or traits instead of parameters – parameters changed for “traits”

Ln 371–376 You write about the results of univariate tests. What about using the multivariate analysis, e.g. canonical discriminant analysis with subsequent classification of individuals into groups (species and/or population and species). – Thank you for your comment, the classification analysis was added, and it helped the study a lot.

Ln 473 B. szaferi - corrected

Ln 490–492 remove capital letters. Morphological variation among Betula nana (diploid), B. pubescens (tetraploid) and their triploid hybrids in Iceland. - corrected

Ln 572 remove “Nonparametric statistics for the behavioral sciences, 2nd ed.” - corrected

Ln 573 replace “Mcgraw-Hill Book Company” by “McGraw-Hill Book Company” - corrected

Ln 574 remove capital letters. Genetical structure of populations - corrected

References

- Complete “doi” where missing, e.g. Järvinen P, et al., 2004; Ln 484 and some other papers. - corrected

- Insert a space after year and semicolon - corrected

- Insert a space after the volume (or remove it), it should be unified - corrected

- Latin names should be in Italics also in References (although this rule is not followed in all papers published in PLoS ONE) - corrected

Reviewer #2

Section Introduction:

I have found that the entire "introduction" section is hard to understand clearly. It is not unequivocally clear which taxa are in question. Also, the literature about the locations of taxa of interest is not clearly stated. The introduction section does not clearly describes the problem related to taxonomic status of the Oyców birch.

We complemented the introduction section to increase its clarity. We defined the research questions and explained the importance of the answers to these questions.

We added some more information to the map in Figure 1, Table. We equipped the manuscript with an additional table in the supplemental information.

Also, in the text of introduction, there is nothing stated about the morphometric and genetic analyses that are used in the research; or the approaches used in research either.

The entire section of the introduction must be revised and supplemented with relevant topics covered in the manuscript.

We have added some more into the Introduction part. However, we think that this may be a topic for the Material and methods section.

Subsection Sampling:

It is required to change subsection name into “Plant material”

- corrected

Line 85: Is the natural origin of the samples obtained from the botanical garden and zoo known?

The specimens in the Chomutov’s ZOO originate in Volyně. These were replanted as young trees from the zone of construction of a high-voltage power line. Information was added to the manuscript.

As for the origin of specimens in Krakow, we addressed the local authorities. If we receive the information soon enough, we will add this information to the manuscript.

Line 108: … although the taxonomic significance of these dark-barked birches remains questioned… This statement does not belong to the section M&M. Move to discussion.

We would prefer leaving the note about taxonomic position here. During the sampling, we have found the dark-barked B. obscura or B. atrata on the surveyed sites. Therefore we decided to include these birches into the analyses, despite the unclarified taxonomic significance of these dark-bared taxa. On the other hand, our study is not aimed at clarification of the taxonomic position of B. obscura or B. atrata. We only want to inform the reader about their inclusion. For this reason, we do not want to open this topic in the discussion section.

Line 117-119: I suggest to the author consider using the term “population”. Terms “working units” and “group” is confusing. If the author still wants to use these terms, he should better explain and define them and not just refer to another reference (line 105-106).

Here we used term “working unit” for birches showing mixed traits between B. pendula and B. oycoviensis. In our opinion, the term “population” is not fully accurate for all situations, as some groups of birches technically distinguished in the manuscript in fact are not populations.

Subsection Morphological analyses:

Line: 131: …. two branches from each sampled individual were collected for analysis…the sentence does not agree with the statement in the line 115-116.

Thank you for your comment, the statement at lines 115 and 116 was corrected; the material for flow cytometry and genetic analyses were both taken from one branch.

Subsection Genome size analysis:

Line: 158: This method measures the amount of DNA per cell (in pg in this case) by comparing the amount of DNA to a known internal standard (in this case Solanum pseudocasicum). Genome size refers to the amount of DNA contained in a haploid genome expressed either in terms of the number of base pairs, kilobases (1 kb = 1000 bp), or megabases (1 Mb = 1 000 000 bp), or as the mass of DNA in picograms (1 pg = 10−12 g)( https://www.sciencedirect.com/topics/immunology-and-microbiology/genome-size ). Please reformulate these sentences with the correct use of the term. This applies to the rest of the text. In general: literature data on the level of ploidy of taxa of interest are mentioned just in the line 214. This information should be stated and commented on in a more transparent way, considering that polyploidization the genus Betula is mentioned.

Thank you for your comment, we have used term “genome size” as in Greilhuber et al. (also cited in the manuscript) and we actually measured the “genome size” in pg (see e.g. Fig 3) and we have used 1Cx values similarly as Greilhuber et al. I think that you mean the same thing by “DNA contained in a haploid genome”, if I get it right.

Subsection Molecular analyses:

Line 179: Which 12 microsatellite markers did you use? Are they listed somewhere? You used the microsatellite markers listed in the literature. However, there must be an exact list of which markers you used.

We have used the same markers as in cited article. We have added the table with markers as supplementary material.

Line 194: You did not determine the "microsatellite length". You determined the length of the amplicons containing the microsatellites. The correct use of the terms is an imperative of scientific work.

Thank you for clarification, the term was corrected.

Section: Statistical analyses and computations/ subsection Morphological analyses:

You did not state how the PCA analysis was performed. How did you use the data given in different units of measurement (mm and degrees of angles) in this analysis? Was the data normalized before PCA analysis? How are they normalized?

How did you analyze continuous and discrete data (e.g. leaf length and nerve number) together in same PCA analysis?

We have removed the PCA analysis and provided new CDA analysis, as it was required by the other reviewer.

Additional requirement

Thank you for including your ethics statement on the online submission form: "Field permit number for sampling and transport of B. humilis:

DZP-WG.6400.6.2020.EP.2 (Generalny dyrektor ochrony Środowiska, Warsaw)

Field permit numbers for sampling and transport of B. oycoviensis:

OP-I.6400.15.2018.KW (Regionalny dyrektor ochrony Środowiska, Krakow) DZP-WG.6400.23.2018.ep (Generalny dyrektor ochrony Środowiska, Warsaw)".

To help ensure that the wording of your manuscript is suitable for publication, would you please also add this statement at the beginning of the Methods section of your manuscript file.

We have added the statement about field permits in the Material and Methods section.

Submitted filename: Response_to_reviewers.docx

Click here for additional data file.

19 Nov 2020

Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: a revised view of the taxonomic treatment of the Ojców birch

PONE-D-20-26980R1

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23 Nov 2020

PONE-D-20-26980R1

Genetic and morphometric variability between populations of Betula ×oycoviensis from Poland and Czechia: a revised view of the taxonomic treatment of the Ojców birch

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