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ISSN 2412-1908
Acta Biologica Sibirica, 2018, 4(2), 47-52
Altai State University
www.asu.ru
RESEARCH ARTICLE
UDC 595.772
Morphometric study of hybridogenic species in Veronica subgenus Pseudolysimachium (Plantaginaceae)
P. Kosachev1, V. Novikova1, S. Pfanzelt2, S. Schongart2, D. Albach2
1Altai State University, Lenina 61, Barnaul, 656049, Russia, E-mail: pakosachev@yandex.ru 2 Carl von Ossietzky-University Oldenburg, Carl von Ossietzky-Str. 9-11, Oldenburg, 26111, Germany
We demonstrate the results of morphometric investigations of hybrids in Veronica subg. Pseudolysimachium (V. * altaica Kosachev und V. * kolyvanensis Kosachev et Shmakov) and their parents. Based on PCoA analysis with seven morphological characters, we reveal an intermediate position of the investigated hybrids and the most important taxonomic characters: ratio of length and width of the lamina of the upper leaves, height of plants, length of the longest corolla lobe and calyx lobe, presence of hairs on the calyx and their position, as well as the length of hairs on the internode below the inflorescence.
Key words: PCoA analysis, hybridization, Veronica spicata and related species, Veronica * altaica, Veronica * kolyvanensis, Altai Mountains, southeastern Europa
Introduction
Härle (1932) suggested that hybridization played an important role in the diversification of the subgenus (formerly variously classified as section or separate genus) and groups of morphological races („Formenkreise") are connected by transitional forms. Later authors (Fischer, 1974; Travnicek, 1998; Albach, Fischer, 2003; Travnicek et al. 2004) partly disagreed and stressed species boundaries, especially based on different types of indumentum (especially presence of glandular hairs). Recent molecular results partly support these species but also emphasized the many transitional forms (Bardy et al., 2011).
Within V. subg. Pseudolysimachium a number of hybrids have been proposed involving V. spicata and its relatives (Härle, 1932; Fischer, 1974; Klokov, 1976; Tsvelev, 1981; Kosachev, 2003, Kosachev, German, 2004; Kosachev, Ebel, 2010; Bardy et al., 2011; Kosachev et al., 2013, 2016, 2017). In the Altai Flora six hybrids are known: V. * altaica Kosachev (V. spicata x V. pinnata), V. * czemalensis Kosachev et Albach (V. porphyriana x V. incana), V. x grisea Kosachev et A.L. Ebel (V. incana x V. longifolia), V. x kolyvanensis Kosachev et Shmakov (V. spicata x V. spuria), V. * schmakoii Kosachev (V. porphyriana x V. longifolia), V. x sessiliflora Bunge (V. porphyriana x V. pinnata).
Kosachev et al. (2016) demonstrated using comparison of DNA sequence data that some of these hybrids indeed involve the proposed parents but for others the complexity of gene flow and introgression between putative parental species and spatial differentiation within these species makes diagnosis of hybrids using molecular tools alone difficult. Therefore, multidimensional comparative investigatioins using several modern methods and data analysis are required to elucidate patterns of ancestry in the putative hybrids.
One of these approaches was followed in an international project to investigate patterns of adaptive introgression in Veronica spicata and relatives involving scientists from three countries: Germany, Ukraine and Russia. The project combines various methods such as molecular genetics (high-throughput sequencing), flow cytometry, ultrastructural analysis of seed and pollen and morphometrics. In the present contribution we present the results of the morphometric analysis of V. spicata and two of its hybrids in the Altai region with the aim to test the hypothesis of hybridization using morphometric analysis and the potential for introgression between parent species via these hybrids.
Materials and Methods
For the investigation of Veronica subg. Pseudolysimachium we used herbarium specimens of recent expeditions (2016, 2017) to the Altai and Europe (Table 1). Voucher specimens are stored in the herbaria ALTB, KW, and OLD.
Kosachev, P. et al. Morphometric study of hybridogenic species in Veronica .... Acta Biologica Sibirica, 2018, 4(2), 47-52 Table 1. Investigated taxa, voucher number and locality_
Species V. pinnata
V. x altaica
V. spicata
Voucher number (ALTB) pin1189
pin1166
pin1162
pin1139
Vouch21
Vouch5_1
alt1140, alt1, alt2, alt4 alt3
spic1127 spic1141, 1144 spic1138 spic1135 spic5U spic12U spic14U spic1H spic2H spic19H
spic21H spic25H
V. x kolyvanensis kol1143, kol1143_2
kol1128, kol1128_2, kol1128 3
V. spuria
spur1142, spur1142_2, spur1142_3 spur1126, spur1126_1
Locality
Russia, Altai Republic, Ongudayskii distr., Aygulak River mouth, h = 1074, 22.07.2016; N 50.35958, E 87.24648
Russia, Altai Republic, Ulaganskii distr., Chuisky Trakt, Chuya River, c. 15 km W of Aktash, N 50,35007, E 87,41245
Russia, Ongudayskii distr., B. Ilgumen River, h = 982 m, 16.07.2016; N 50.64244, E 86.36352
Russia, Altaiskii krai, Tretjakovskii distr., above Aley River, h = 394 m, 13.07.2016; N 50.91591, E 82.32704
Russia, Republic Altai, Ulaganskii distr., 8 km SSE from village Aktash, River valley, H = 1491,5 m, steppe, N 50.344583, E 87.441745 Russia, Republic Altai, Ulaganskii distr., Chulyshman River, H = 627,8 m, steppe, N 50°58'39", E 88°05' 24,8"
Russia, Altaiskii krai, Tretjakovskii distr., above Aley River, h = 394 m, 13.07.2016; N 50.91591, E 82.32704
Russia, Altaiskii krai, Tretjakovskii distr., mount Poruczikova, village Ekaterininskoe, N 50°54'30.8", E 82°00'56.6"E Russia, Altaiskii krai, Kurjinskii distr., 15 km N of Kurja, h=213 m, 12.07.2016; N 51.76835, E 82.13831
Russia, Altaiskii krai, Kurjinskii distr., Loktewka River, h = 423 m, 14.07.2016; 51.29644 N, 82.49078 E
Russia, Altaiskii krai, Tretjakovskii distr., above Aley River, h = 394 m, 13.07.2016; N 50.91591, E 82.32704 Russia, Altaiskii krai, Zmeinogorskii distr., 3 km N of Lake Kolyvanskoye, N 51.39235, E 82.20837
Ukraine, Oblast Kiew: Koccha zaspa meadows, grassland, 19.5 m from creek margin, h = 92 m; N 50.329417 E 30.578733 Ukraine, Oblast Chmelnyzkyj: Four-Horseman, near Verbka, meadows, low grass, h = 252 m; N 48.801483, E 26.598317 Ukraine, Oblast Tscherniwzi: between Shershenivka and Oleksyntsi, meadows, grass, rocks, h = 206 m; N 48.817200, E 25.829733 Hungary, Tar-Ko, Bukk NP, cliffs, h = 950 m; N 48°02'21.72", E 20°27'39.96"
Hungary, Borsod-Abauj-Zemplen: Pereces, tall grass meadow above orchard, h = 250 m; N 48°07'49.96", E 20°40'54.50" Hungary, Eisenburg: Sarvar, 47°17'02.76" N 17°02'22.62" E, abandoned vineyard, on basalt, h = 186 m; N 47°17'02.76", E 17°02'22.62"
Hungary, Sar-Hegy, Gyongyos, open vineyard, h = 280 m; N 47°48'20.74", E 19°59'30.96"
Hungary, Eisenburg: Tokorcs, dry meadow, h = 190 m; N 47°17'37.98", E 17°06'15.24"
Russia, Altaiskii krai, Kur'inskii distr., Loktewka River, h = 423 m, 14.07.2016; N 51.29644, E 82.49078
Russia, Altaiskii krai, Kur'inskii distr., along roadside (K9) between Pospelikha and Kurya (c. 20 km NNW of Kurya), N 51.76835, E 82,13832
Russia, Altaiskii krai, Kur'inskii distr., Loktewka River, h = 423 m, 14.07.2016; N 51.29644, E 82.49078
Russia, Altaiskii krai, Kur'inskii distr., along roadside (K9) between Pospelikha and Kurya (c. 20 km NNW of Kurya) N 51.76835, E 82,13832
Here, we investigated two hybridogenic taxa V. * altaica with its parents V. spicata and V. pinnata; V. * kolyvanensis with its parents V. spicata and V. spuria. For the study we used 33 herbarium specimens from 24 populations. For all taxa, at least 10 plants were scored and all characters measured at least five times. All measurements were conducted under a light microscope Olympus BX 51 with the software ZEN (Carl Zeiss).
Quantitative measurements were conducted on the basis of the study by Bardy et al. (2011). The authors of this study initially investigated 18 potentially informative morphological characters (1 qualitative, 17 quantitative characters)
based on suggestions by Albach & Fischer (2003). Nine of these characters were shown to be correlated and excluded by Bardy et al. (2011).
In our study we used the following important, diagnostic characters (Table 2): Two characters - percentage of stalked glandular hairs on sepals and percentage of sessile glandular hairs on sepals - were not used due to large error in measurements. However, we have adapted these characters to a further qualitative character, the presence of glandular hairs on sepals. If present, we have also noted the distribution (ciliate vs. Present also on the surface).
The data matrix was converted in a distance matrix using the Gower-coefficient for a PCoA. All analyses were conducted using R v. 2.10.1 (https://cran-archive.r-project.org/bin/windows/base/old/2.10.1/).
Table 2: Morphological characters used in the analysis with their abbreviation
Abbreviation Morphological character
LLP Length of the longest petal
WC Width of corolla
LLS Length of the longest sepal
LWL Length / width of lamina of the leaf pair below the one subtending the inflorescence
PH Total plant height
SHL Length of hairs on stem on the internodium below the inflorescence
Absence (1) or presence (2 / 3) of hairs on the calyx. If present, hairs were score: (2) simple 3-
4-celled hairs on the margin and 1 -2-celled glandular hairs on the surface; (3) only 1 -2-celled
GH glandular hairs distributed all over the sepal
Results
The analysis of principal coordinates demonstrate almost complete separation of the taxa using our seven characters. Figure 1 depicts the situation for V. * altaica and its putative parents.
PCoA biplot Response variables projected as in PCA with scaling 1
-1
o
1
pin1189
pin1166
3IC21H spiel 141 . spid 127 \ LLS \ spid 9H ^ \
spid 144 ^--jf^v Spic2 m^m^ysS11 „u ; /aH140 G|-fepicaf>ic2bfalt1 spid 2U spid H
SHL
-0 4
-02
00
02
04
0.6
Axis.1
Fig. 1. Principal Co-ordinate Analysis of a matrix of pair-wise Gower distances based on 7 morphological characters scored for 24 individuals from 20 populations of the Veronica * altaica and its putative parents V. spicata and V. pinnata from weastern Altai Mountains and southeastern Europa
Veronica pinnata is clearly separated from all other taxa along axis 1 on the basis of ratio length to width of lamina. Additionally, populations pin 1189 and pin1166, growing close to each other in the Chuya valley, are separated from other populations of the species due to differences in corolla size.
V. x altaica is separated from the putative parent V. pinnata by all characters but not from its other putative parent, V. spicata. However, mixture occurs only through the Hungarian population spic1H through the character hair length on the internode below the inflorescence. Veronica x altaica differs from all other populations, including the sympatric ones, in this character.
V. spicata is diverse in our measured characters and no differentiation between populations, e.g. between European and Asian populations is discernable. Least variation is detected in width of the corolla and length of longest sepal.
Figure 2 depicts the PCoA for V. x kolyvanensis.
All three taxa are markedly differentiated from each other. On axis 1, V. spuria is separated markedly from the other taxa by the character height of plants. For V. x kolyvanensis height of plants and length of longest corolla lobe are positively correlated. Population spic1127, which is sympatric with kol1128 in Kur'inskii region of the Altaiiskii krai, is closest to V. x kolyvanensis, especially due to length of longest corolla lobe but also all other characters. Noteworthy is some separation between Asian and European populations of V. spicata using characters SHL, WC and GH (hair length on stem, corolla width, calyx indumentum).
PCoA biplot Response variables projected as in PCA with scaling 1
SHL
LWl spid 2U spic2H
Ml42-2 sPmm PH < spic25H spic21 H ,.,„ spic5U wc GH spiel 9H 4U
spurl 142 LLP spiel
kol1143 spiel 127
kol1143_
MejmtfflS koM128
-0.4
-0.2
0.0
0.2
0.4
Axis.1
Fig. 2. Principal Co-ordinate Analysis of a matrix of pair-wise Gower distances based on 7 morphological characters scored for 25 individuals from 16 populations of the Veronica x kolyvanensis and its putative parents V. spicata and V. spuria from weastern Altai Mountains and southeastern Europa
Discussion
In both cases can we recognize a clear separation of the taxa. This may partly due to putative parents being in different subsections of V. subg. Pseudolysimachium with clear morphological distinction. Another part of the explanation is that we chose examples with hybrids markedly differing from its parents to prove that our approach works in cases of a priori distinct hybrids.
In both cases, hybrid taxa take on an intermediate position between the parents. Special importance in this respect have characters such as ratio of length and width of leaf lamina (V. x altaica) and height of plants (V. x kolyvanensis).
Populations of V. pinnata are well characterized by the ratio of length to width of the lamina in upper leaves. Leaves are linear and much longer than wide. In general, the species is easily distinguished by the pinnatifid leaves with linear segments and set apart in Sect. Pinnatae (Holub) Kosachev et Albach (Subg. Pseudolysimachium). Populations from the Chuva valley (pin1189, pin1166) are distinguished from other populations by longer corolla lobes, approaching V. spicata
and V. porphyriana. It is possible that introgressive hybridization with V. porphyriana, which is only sympatric with V. pinnata in the Western Altai, plays a role. Here, the hybrid of both species, V. * sessiliflora, has been described.
Populations of V. * altaica resemble V. spicata in ratio of leaf length to width, presence of glandular hairs on the sepals and height of plants. Preliminary analyses of NGS data suggest that V. * altaica serves mainly as a way of introgression towards V. spicata (Felgentreu & Albach, unpubl.). However, alternative scenarios will need to be investigated based on differences between V. * altaica and both its parents. For example, V. * altaica and V. spicata differ in the Altai in length of hairs on stem below inflorescence with V. * altaica having 1.5-2x longer hairs than V. spicata. It is not clear whether this character is transgressive or hints at hybridization with a third species. A sample of V. spicata from Hungary, which we identified as V. spicata ssp. fischeri, has been noted to have even longer hairs, 2x longer than V. * altaica and 3-4x longer hairs than V. spicata in the Altai. Whether this is due to mutations in this subspecies or due to introgression from yet another taxon (e.g., V. incana) will also need to be further investigated. Bardy et al. (2011) provided some evidence for the latter. If a transgressive elongation of hairs in hybrids is possible in V. * altaica, it may, however, also be possible in V. spicata ssp. fischeri without introgression from V. incana. Since apart from hair length, corolla width and calyx indumentum differ between populations of V. spicata in Europe and the Altai, and we also see this in DNA sequence analysis (Felgentreu & Albach, unpubl.), the differentiation in V. spicata will definitely need further analysis.
V. * kolyvanensis is intermediate between its putative parents V. spicata and V. spuria in height of plants and length of longest corolla lobe. Specimens of V. spicata sympatric with V. * kolyvanensis samples (spic1127, spic1141) approach V. * kolyvanensis, again suggesting possible introgression. Other characters demonstrate a mixture in V. * kolyvanensis, such as indumentum on the sepals, which comprises short glandular hairs on the surface, resembling V. spicata, and also the margin, where V. spicata has long cilia but similar to V. spuria, which in turn has glabrous sepal surface. The latter species is clearly distinct from the other studied species based on height of plants, glabrous sepal surface and short corolla and calyx lobes.
Finally, we found decisive differences between European V. spicata and specimens from the Altai in length of hairs on stem on the internodium below the inflorescence and on the sepals. Future genetic analyses, therefore should consider introgression between V. incana and V. spicata in Europe.
In summary, we have demonstrated that hybrids in V. subg. Pseudolysimachium are distinguishable from its parents using morphological analysis. The most important characters in this respect are ratio of leaf length to leaf width, height of plants, length of hairs, as well as its distribution on its calyx. Furthermore, morphometrics is also able to provide initial hypotheses for introgression between species and their hybrids. These hypotheses will need to be further analyzed using comparative morphological and molecular analyses.
Acknowledgments
The study was supported by the Volkswagen-Foundation, project 90256.
References
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Citation:
Kosachev, P., Novikova, V., Pfanzelt, S., Schongart, S., Albach, D. (2018). Morphometric study of hybridogenic species in Veronica subgenus Pseudolysimachium (Plantaginaceae). Acta Biologica Sibirica, 4 (2), 47—52. Submitted: 13.02.2018. Accepted: 25.04.2018 erossrcflittp://dx.doi.org/10.14258/abs.v4i2.4123
© 2018 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
