METHOD OF FIELD AND COMPUTER MORPHOMETRY IN DIAGNOSTICS OF
DIPHASIASTRUM (LYCOPODIACEAE S.L.) TAXA IN THE MOSCOW REGION
*Vitaly M. Еfanov, Natalia A. Guseva, Anna G. Bega
State Educational Institution, Moscow, Russian Federation
Moscow State Regional University, Moscow, Russian Federation
Abstract: The article deals with the topic of species diagnosis in the genus Diphasiastrum. At the moment, all the methods available to researchers require the removal of whole plant samples or parts of plants from populations. In this paper we propose a new express method for obtaining morphometric data of species of the genus Diphasiastrum without plant damage and give its hardware implementation. The method allows the identification of species directly in the field. Field and desktop testing of the proposed method was carried out. The low variance of repeated measurements of species-specific traits suggests that the method is suitable. Three new locations of Diphasiastrum in the Moscow region have been discovered and described. The plants were described according to a set of diagnostically relevant qualitative and quantitative indicators and identified as D. complanatum (L.) Holub subsp. complanatum. For each finding, a standard description of the biotope is given and a conclusion is made as to the degree to which the location is potentially suitable for the long-term existence of the species population. We consider the proposed method to be potentially suitable for many members of the Lycopodiaceae family. The relative rarity of many of these plants in nature makes the appearance of the method timely.
Keywords: species identification, morphometry, plants conservation, Diphasiastrum.
Running title: V.M. Efanov et al. The method of field morphometry in the diagnostics of Diphasiastrum
Different approaches and methods have been used to decide on the species rank of the clubmosses taxon. This has sometimes led to contradictory interpretations and to the complication of synonymy. In our view, the best solution to this problem should be to adopt a conventional approach [Evo J.S., 2016]. A combination of methods, ranging from in-depth morphological analysis to molecular diagnostics, is desirable to improve the accuracy of direct detection [Stoor A.M. et all., 1996; Horn K. et.all., 2006; Bennert W. et.all., 2011; Klein L.L., 2012; Szypula W.J., 2013]. This will improve credibility through an independent method of identification. In this article we propose a field method for obtaining morphometric data for species of the genus Diphasiastrum. At the moment, all existing methods of identification of clubmosses are associated with an increase in anthropogenic impacts on wild populations. Morphological analysis involves collecting living plants or plant parts for cameral morphometry (herbarium and fixed sporophyte specimens). In our opinion, all of the clubmosses in the Moscow region are in need of increased protection. The use of non‑destructive methods of investigation has already been raised in a number of papers [Panchenko S.M., 2000; 2009; Panchenko S.M. and Chernous О.P., 2005;] on the vitality analysis of clubmosses. However, the authors were not able to exclude phylloid harvesting and only managed to reduce it to 28-32 specimens from orthostichia, which, by definition, is not a nondestructive action proper and can be dangerous because of the probable rotting of the damaged part of the shoot [Benca J.P., 2020]. There are also separatepapers that note the need to analyse lifetime images of plants, but do not propose field morphometric methods themselves [Bjork C.R., 2020].
Diphasiastrum Holub, 1975 – isolated by the Czech botanist Josef Holub in 1975 during the revision of Lycopodium s.l., is considered a relatively young, Cenozoic genus of clubmosses [Schnittler M. et all., 2019]. Diphasiastrum is distinguished from other Lycopodium s.l. by flattened (in most species) lateral vegetative branches of orthotropic shoot systems with a decussate arrangement of scale-like phylloids, radical gametophyte form (so-called “complanatum-type”) and a chromosome number unique among the clubmosses genera, 2n=46 [Holub J., 1975].
The species Diphasiastrum complanatum (L.) Holub and D. tristachyum (Pursh) Holub occurring in the Moscow area have a differentiated above-ground shoot system with a well-defined main axis and fan‑like systems of dichotomous lateral branches. The general habitus of the orthotropic shoot systems of these species is described as tree-like by the genus Joan Wils [Wilce J.H., 1965]. The life form of Diphasiastrum species can be described as bushy, as these plants are characterised by low perennial systems of above-ground shoots [Ivanenko Y.А., 2016]. The phylloids of Diphasiastrum can be iso-, di- or trimorphic, which is related to the degree of flattening of the twigs. Plagiotropic shoots are creeping or crawling and can be located at different substrate depths, allowing them to be considered functionally as epi- and hypogeogenic. Deeper in the substratum, hypogeogenic shoots are paler in colour, but green when they come above the ground and are capable of surviving forest fires. Strobili is on strobilus-bearing peduncle, less often sessile, and the species near Moscow have a peduncle of varying degrees of branching [Ivanenko Y.А., 2016; 2004; 2013].
The genus Diphasiastrum found in different latitudinal and vertical zoning belts. In sympatric zones, interspecific hybridisation is known. For example, in Central Europe, three parental and three hybridogenic species are distributed sympatrically [Bennert W. et all., 2011; Schnittler M. et all., 2019; Hanusova K. et all., 2014]. The ecological niches of Diphasiastrum species of the Moscow region partially overlap. D. tristachyum inhabits drier and brighter habitats, whereas D. complanatum can also be found in more shaded, wetter habitats, and the hybridogenic D. x zeilleri (Rouy) Holub, with the parent formula D. tristachyum x D. complanatum, occupies an intermediate position both morphologically and ecologically [Ivanenko Y.А., 2016; 2004]. Diphasiastrum is thus a very interesting subject for sistemic‑biological research. However, in terms of species identification, Diphasiastrum is a complex group that requires consideration of many diagnostic features. The International Pteridophyte Phylogeny Group recognises the genus Diphasiastrum as monophyletic, comprising 20 species [Evo J.S., 2016]. Some taxa are treated as hybridogenic species [Ivanenko Y.А., 2016; 2004; 2013], which is also widely recognized as a scientific community [World Flora Online 2000].
The aforementioned features of Diphasiastrum structure, such as the presence of fairly compact systems of orthotropic shoots with often prostrate, flattened lateral branches bearing polymorphic decussate phylloids, allowing easy survey of diagnostically significant morphological characters in the field, led to the choice of this genus as an object for testing a new method of field morphometry.
The aim of our work was to create a rapidin vivo method for obtaining a set of diagnostically relevant morphometric data of representatives of the genus Diphasiastrum. To this end, the following objectives were set:
- Develop an algorithm for field and cameral studies of clubmosses, and propose a hardware implementation of the method.
- Conduct a field test of the method when searching for new locations of Diphasiastrum.
- Evaluate the applicability of the method by comparing data collected under different modusoperandi of obtaining samples. Suggest possibilities for prospective improvements to the method.
Materials and methods. This paper proposes a method for collecting morphometric data of Diphasiastrum without plant damage, based on photography in the field. The method is designed to evaluate the features used in the single‑input dichotomous textual [Sviridov А.V., 2012] keys proposed by Y.A. Ivanenko and N.N. Tzvelev [Ivanenko Y.А. and Tzvelev N.N. 2004]. We have designed and manufactured a model of a field measurement device, the Field Morphometer (FM). The unit consists of three functional parts: the base with the measuring scale, the mounting feet and the clamping device (pic. 1).
The morphometer made it possible to attach plant parts easily, without damaging them, to capture morphological features. The morphometer algorithm consisted of the following steps: the instrument was fixed in the soil by selecting a suitable angle for the mounting feet, and sections of the partial shrub with well-developed branches that had finished their growth and branches with current year’s increment, were selected for measurement. The plants were attached to the FM and then photographed together with a 13.0 megapixel camera scale with a resolution of 4208×3120 pixels. Particular attention was paid to filming the ventral side of the branches of orthotropic shoot systems.
In addition to obtaining photographic images, scanned images were obtained for each clonal colony if possible. To obtain them, senile and dead parts of orthotropic shoot and branch systems meeting the conditions given above were collected. The material was labelled and immediately placed for transport in a herbarium folder or a tightly closed container. Scanned images were obtained by digitising the collected material together with the scale on a Brother DCP-7055R MFP at maximum resolution (1200×1200 dpi), the image format being uncompressed tif.
In the cameral phase, ImageJ v.1.52n (Oracle Java v.1.7.8 32-bit) was used as the software component of the method. The measuring scale was calibrated before working with each sample [Коnyukhov, 2012]. If necessary, the images were sharpened using an appropriate filter in Paint.Net v.3.5.11. Each sample was measured in quadruplicate. The measurements were taken on developed parts of the shoots that were not susceptible to rotting. The linear measurement data obtained in ImageJ were entered into Apache Open Office Calc v.4.1.8 worksheets and the average values were calculated (Table 1).
The description of the colonies included a set of qualitative indicators: habitus, nature of formation of fertile structures: presence of strobils and length of peduncle, degree of peduncle branching and separation from the fertile branch, number of strobils on the peduncle and presence or absence of peduncle branches, colour of plagiotropic shoots, depth and nature of their occurrence. The flattening of lateral branches of orthotropic shoot systems and their colour on the developed part of the branch and in the areas of annual growth, if any, were visually assessed.
Picture 1. Field Morphometer (FM). Explanation. A – base with measuring scale; B – mounting feet; C – general view of the device (scheme); D – FM with sample in working position, thin, narrow rings of transparent latex are used for fixation of plants.
Table 1. Morphometric data of the Diphasiastrum specimens examined
|Free part of the
width w, (mm)
|m15XI2020Еc1s1||2, 99||0, 56||1, 15||5, 36||0, 54||1, 13||5, 65|
|m15XI2020Еc1s2||3, 13||0, 53||1, 10||5, 95|
|m15XI2020Еc2s1||3, 08||0, 51||0, 96||5, 99||0, 51||1, 11||5, 83|
|m15XI2020Еc2s2||3, 55||0, 60||1, 25||5, 96|
|m15XI2020Еc2s3#||2, 95||0, 49||1, 24||5, 97|
|m15XI2020Еc2s4#||2, 45||0, 45||1, 01||5, 40|
|m29VII2020Sc1s1||4, 50||0, 69||1, 23||6, 50||0, 57||1, 03||6, 86|
|m29VII2020Sc1s2||4, 12||0, 55||1, 01||7, 54|
|m29VII2020Sc1s3||3, 54||0, 51||0, 98||6, 99|
|m29VII2020Sc1s4||3, 36||0, 52||0, 91||6, 42|
|m29VII2020Sc2s1||3, 07||0, 54||0, 99||5, 70||0, 51||1, 08||6, 11|
|m29VII2020Sc2s2||3, 57||0, 48||0, 96||7, 39|
|m29VII2020Sc2s3||3, 08||0, 55||1, 27||5, 61|
|m29VII2020Sc2s4#||2, 86||0, 50||1, 08||5, 74|
|m29VII2020Sc2s5#||3, 05||0, 50||1, 09||6, 13|
|m22III2020Sc1s1#||2, 82||0, 47||1, 15||5, 96||0, 48||1, 15||5, 90|
|m22III2020Shc1s2#||2, 88||0, 49||1, 15||5, 84|
Notes: m – morphometric sample, # – scanned sample, 15XI2020 – date of collection, E – Yegoryevsk district, S – Stupino district, Sh – Shatura district, c1 – colony number, s1- sample number.
As quantitative indicators, the width of lateral branches of orthotropic shoot systems (W); the base width of the free part of the ventral phylloid (w) on the developed part of the branch (2 measurements) and in the current year’s growth zone (2 measurements); and the length of the free part of the ventral phylloid. The W value, taking into account the free part of the lateral phylloids, was measured in the area of their maximum width by placing the ends of the measuring line near the middle of the free parts of the lateral phylloids, orienting the line perpendicular to the branch axis.
Measurements of the base width and free length of the ventral phylloids were made in a similar manner, orienting the measurement line perpendicular to the organ axis in the first case and parallel in the second (Fig. 2A). As the most diagnostically valuable feature, the ratio of the width of the lateral branches of orthotropic shoot systems to the width of the ventral phylloid base (W/w), measured as described above, was taken (with due consideration of the others). This feature, is given in definitional keys [Ivanenko and Tzvelev 2004] and helps to distinguish parental species of Diphasiastrum.
To test the applicability of the method, digital specimens from the Syreyschikov Moscow University Herbarium, Faculty of Biology, Moscow State University (MW) [Seregin, 2021], assigned to D. tristachyum, D. complanatum and D. x zeilleri, were processed. One sample of each species, originating from the Moscow region and with an informative label, was chosen. When processing these samples, only the W values were recorded, as the low resolution of these samples prevented the remaining measurements from being taken with comparable accuracy. Each sample of any type was assigned a unique code (matching the sample file name or MW code), including information on the date, location, type and number of the sample. All measurements of the sample were assigned a number from 1 to 12.
This detailed coding of objects allows you to go back to any criticised measurement at the right moment and double-check it.
During the field assay, the plants were searched using the route method [Artayev, et all., 2014] and a biotope description was prepared for each find [Kharitonov N.P., 1998]. As it is difficult to establish the exact boundaries of the colonies without harming the plants, a colony at least 300 metres away was treated as a separate colony, otherwise it was taken as a clonal particula of the colony in question. A total of three locations of Diphasiastrum from Shatura, Stupino and Yegoryevsk districts of Moscow Region were found and described, and 11 photographic and nine scanned images were analysed (pic. 2B-F).
Picture 2. Computer morphometry of Diphasiastrum. Explanation. A – localization of measurements: 1 – width of lateral branch of orthotropic shoot systems (W); 2 – base width of ventral phylloid free part (w); 3 – length of ventral phylloid free part. B – some measurements of sample m29VII2020Sc1s1; C – also, sample m15XI2020Ec2s2; D – sample of poor preservation m22III2020Shc1s2#; E – sample m29VII2020Sc2s5#; F – fragment of herbarium specimen MHA0032405.
Results and discussion. In the course of our fieldwork we discovered three new locations of Diphasiastrum in Shatura, Stupino and Yegoryevsk districts of Moscow Region, where geobotanical descriptions were made and studies of Diphasiastrum colonies were carried out.
In Shatura District Diphasiastrum sp. was discovered on 22.03.2020 half a km east of Pozhoga village (55°25’24.6 “N, 39°42’35.9 “E). The site is located in the south-western part of the Meshchera lowlands, the terrain is poorly rugged, mostly flat, and the soils are sod-podzolic, on sands.
A single colony of Diphasiastrum was found on the edge of a recent windfall in a lightened (30-35% crown cover) pine forest. Pinussylvestris L., Picea abies (L.) H.Karst., Betula pendula Roth, less frequently Sorbus aucuparia L. and Quercus robur L., in the shrub layer – Rubus idaeus L., Juniperus communis L., Lonicera xylosteum L., Frangula alnus Mill. and occasionally Daphnemezereum L. Grass-bush layer includes Callunavulgaris (L.) Hull, Convallariamajalis L., Vacciniummyrtillus L., V. vitis-idaea L., Asarumeuropaeum L., Fragariavesca L., Stellariaholostea L., Pteridiumaquilinum (L.) Kuhn, Rubussaxatilis L., Luzulapilosa (L.) Willd., Oxalisacetosella L., Calamagrostisepigeios (L.) Roth. and Carexericetorum Pollich. The shrub and herb‑shrub tiers are not always equally expressed here; in the latter, the projective coverage of background species (Vacciniummyrtillus, Calamagrostisepigeios, Carexericetorum, Callunavulgaris) is uneven, which leaves free green-mossy areas with rare sandy outcrops on windfalls. The moss-lichen layer is composed of Dicranum sp., Pleuroziumschreberi (Willd. ex Brid.) Mitt., Climaciumdendroides (Hedw.) F. Weber & D.Mohr, Polytrichumcommune Hedw., Atrichumundulatum (Hedw.) P. Beauv. and in wet depressions, Sphagnum sp. and Mnium sp.
The small (0.75-1 m2) colony (m22III2020Shc1s1-2#) is located in a green-mossy biotope with small participation of shrub, grass-bush and undergrowth. Orthotropic shoots are intensively branched, spreading, 11-15 cm high, their lateral branches flattened, deep green on the dorsal side, including shoots from the previous growing season, ventral side light green, current year shoots not expressed. The plagiotropic shoots are greenish-white or light green, epigeogenic. Senile shoots in insignificant numbers, found 4 last year’s strobils sitting alone on a double-branched (4.5-5 cm) peduncle, clearly detached from the lateral fertile branch of the orthotropic shoot. Two other species of the family, Lycopodiumclavatum L. and Spinulumannotinum (L.) A. Haines, of which the former occurs most frequently, were also found here.
Location from 29.VII.2020 two kilometres south-west of Belopesotsky station (54°51’16, 2”N, 38°06’56, 2”E), Stupino district, located in south-eastern part of Moskvoretsko-Oka plain, in flood plain mixed pine forests between river Kremnitsa and Oka. The terrain is rugged, hilly, with sparse natural outcrops, and the landscape has been heavily transformed by anthropogenic activity. Soils are grey forest loamy, sandy loam and sod-podzolic, on sands. Two large colonies of Diphasiastrum were found, the first containing two and the second four clonal particulas.
The first of the colonies (m29VII2020 Sc1s1-4) occupies the top and northeast hillside in a dry biotope (55-60% crown cover), in a hilly area. Betulapendula and Pinussylvestris, less often Piceaabies, Quercusrobur and Sorbusaucuparia, Frangulaalnus, Juniperuscommunis, Euonymusverrucosus Scop, Loniceraxylosteum and Rubusidaeus, in herb-shrub layer: Stellariaholostea, Callunavulgaris, Melampyrumnemorosum Baumg., Ajugareptans L., Vacciniumvitis-idaea and V.myrtillus, Pyrolarotundifolia L., Pteridiumaquilinum, Oxalisacetosella, Asarumeuropaeum, Calamagrostisepigeios, Luzulapilosa and Carexpallescens L.As in the previous location, the projective cover of the background species of the grass-bush layer is uneven and the colonies found here are confined to areas with less participation of marginal grassland species such as Calamagrostisepigeios and Carexpallescens. The moss-lichen layer is dominated by Pleuroziumschreberi, Dicranum sp. and Polytrichumcommune, with Climaciumdendroides and Atrichumundulatum, Rhodobryumroseum (Hedw.) Limpr. in wetter depressions, and Polytrichumjuniperinum Hedw., P. piliferum Hedw. and Cladonia sp. on dry sandy outcrops. The colony consists of two clonal particulas measuring 6.5 x 6 and 2 x 2.5 m, spaced a few metres apart. Intensively branched orthotropic shoots systems prostrate, 12-16 cm tall, lateral branches flattened, dark green dorsally, light green ventral side, lettuce‑green shoots. The plagiotrophic shoots are epigeogenic, located in moss cushions and/or leaf litter, greenish-white to green. The senile parts of the shoots are relatively evenly distributed over the colony area. The smaller of the clonal particula has no strobili, the larger has a high (over 200) number of strobili, 2-5 arranged on long, separated from the lateral fertile branches peduncles, and a single case of strobilus dichotomy was observed.
More than half a kilometre north-east of the first colony, a second colony (m29VII2020Sc2s1-5), even larger, consisting of four clonal particulas, was found. The colony is located in a sloping somewhat more shaded and moist, green-mossy depression of the same biotope, with low projective cover of the background species of the herbaceous-bush layer. In terms of external diagnostic features, the plants of the second colony correspond to those of the first colony. The presence of strobili was noted in half of the second colony particulas.
In Yegoryevsk District, Diphasiastrum sp. was found 15.XI.2020, one kilometre northeast of Rudnikovskaya Station, (m15XI2020Ec1-2s1-4) less than one kilometre north of the Lopatinsky Phosphorite Mine (55°20’31.2″ N 38°55’31.5″ E), located in the southwest part of the Meschera lowlands. This Diphasiastrum habitat is of a distinctly secondary nature and is located in medium-aged regenerative mono‑planting of Pinussylvestris, on sandy, weakly podzolized soils, probably on the site of former geological workings. The thickness of the upper horizons of the soil profile here is extremely low (>>10 cm), resulting in oligotrophic conditions, along with the landslide nature of some sections of the bank of a large marshy ravine, which skirts this location in a semicircle. With moderate (40-50%) crown cover, Pinussylvestris and Piceaabies are rare in the undergrowth, while the admixture of deciduous species is negligible, sometimes Sorbusaucuparia and even more rarely Betulapendula.The shrub tier is almost not expressed and represented mainly by Juniperuscommunis, much less often Loniceraxylosteum and Fragulaalnus. The herb-shrub tier is poorly developed and is represented by Antennariadioica (L.) Gaertn. (in some places in mass), Callunavulgaris, Vacciniummyrtillus, less frequently V. vitis–idaea and Luzulapilosa, Calamagrostisepigeios (single) and Carexpallescens. Two other species of clubmosses, Lycopodiumclavatum and Spinulumannotinum, have been recorded as part of the herb-bush layer. Both species grow explerentially, with high levels of projective cover. Lycopodiumclavatum predominates. Moss‑lichen layer of Dicranum sp., Polytrichumjuniperinum, P. commune and P. piliferum, Climaciumdendroides, Pleuroziumschreberi, Cladonia sp. and Cetrariaislandica (L.) Ach. Due to the oligotrophic conditions and the low development of herbaceous-shrub layer, the biotope is characterised by a low level of competitive pressure. Here, two colonies of Diphasiastrum were found at a sufficient distance, the plants of which appeared to be similar in terms of a set of external diagnostic features. Lateral branches of orthotropic shoots broad, strongly flattened, ventral side light, lettuce-green, dorsal side dark green, without a bluish‑grey tint. Plagiotropic shoots of epigeogenic type, greenish-white to greenish colouration. No strobili were found.
The morphometric data of all samples are shown in Table 1. In the course of cameral processing of the materials obtained, we found that plants from all three locations described above can be assigned to the nominal subspecies of D. complanatum (L.) Holub subsp. complanatum. All specimens examined have fairly broad and flattened, fan-shaped lateral branches of orthotropic shoot systems with broad lateral and reduced, narrow, often styloid ventral phylloids. This set of features has a low (less than 20%) coefficient of variation, suggesting low variability of the featuresand their high diagnostic significance. The width of lateral branches of orthotropic shoot systems is the most variable, while the width of the base of the free part of the ventral phylloid is the least variable (Table 2). The average values obtained for the base width of the free part and the length of the ventral phylloid proved to be fairly constant consistently for samples of each colony and location. The diagnostic value W/w depends on the accuracy of the measurement of the base width of the free ventral phylloid (w).
The morphometric data of the samples we collected agree with the values of the diagnostic keys used. Some deviations into the area of larger mean branch widths are present for the Stupino district (m29VII2020Sc1-2s1-4-5). However, the average measurements of the ventral phylloid of these specimens are consistent with those from other locations, with its slightly shorter average length. From a systems‑biological perspective, one explanation for this deviation could be the relatively lower light conditions. The large admixture of deciduous species in the stand at this location and the significant development of undergrowth are consistent with the observed greater branching width and reduction of the ventral phylloid. However, such a pattern must be proven on statistically significant material. In order to obtain objective data on light levels, we consider it promising to use the luxmeter in further system‑biological studies.
The open-source program ImageJ was chosen because of its cross-platform nature, low resource requirements, extensibility and automatability [Коnyukhov, 2012; Mitsik, 2011].
Table 2. Statistical indicators of the sample
|Indicator||Yegoryevsk district||Stupino district||Shatura district||The whole sample|
|coefficient of variation
of the indicator (%)
Notes: W – width of lateral branches of orthotropic shoot systems, w – width of the free part of the ventral phylloid, l-length of the free part of the ventral phylloid
The applicability of the method depends directly on the quality of the images being processed. Thus, analysis and verification of our photographic and scanned images is not difficult, whereas for MW specimens this was not always possible due to insufficient scanning resolution (Fig. 2F) and the peculiarities of the herbarization of individual specimens. The lateral branch width (W) of the orthotropic shoot systems of the MW samples falls within the range of reference values of the diagnostic keys used [Ivanenko and Svelyev 2004]. However, while specimens MW0208486 and MW0208335 that we examined are informative on the entire complex of external, and on part of the traits available for measurement, for MHA0032405 it was impossible to evaluate the entire diagnostic complex given in the special literature [Ivanenko Y.А., 2016; 2004; 2013], as the specimen has no strobili and fully preserved plagiotropic areas at all. For this specimen, only the available part of the morphometric data, the branching pattern and the shading of the lateral branches of the orthotropic shoot systems were evaluated. We consider it mandatory to check the location of MHA0032405 in order to clarify the diagnosis, the identification of other MW samples is confirmed by us comprehensively.
From our point of view, we characterise the Diphasiastrum occurrences described here differently for the purposes of phytodiversity protection. The Shatura district location is of less concern due to its low population density and the presence of suitable plant microhabitats, as evidenced by the combined occurrence of the three species of the family. The biggest concern is the find from the Stupino district. Despite the large size of the colonies and the potential abundance of spore production, plants at this location are clearly at risk of extinction due to high recreational pressure, signs of which were observed along the entire route. The absence of other clubmosses species and the observed successional changes in this section of the Oka floodplain indigenous woodland confirm our fears. Despite indications of D. complanatum at other locations in the Stupino and Serpukhov districts [Bega and Yefanov, 2018], we note that some of them are not confirmed, and the repeated searches we have undertaken since 2017 have shown a high level of recreational pressure and increased succession in many parts of the south of the Moscow region. The find from the Yegoryevsk district is interesting because it shows the high ability of the plants to successfully develop suitable secondary habitats. Nevertheless, secondary habitats, as opposed to the increasingly rare primary habitats, cannot be considered a reliable reservoir of clubmosses populations.
The method proposed in this paper has produced photographic images that are as informative as herbarium specimens (“in vivo herbarisation”). The hardware implementation of the proposed method was successfully tested in the description of three Diphasiastrum locations in the Moscow region. Thus, it can be concluded that it is suitable for obtaining diagnostically relevant information on the Diphasiastrum species complex. The method has a number of positive characteristics: high accuracy, limited only by technical equipment; ease of use and versatility, expressed in the adaptability of the morphometric protocol. The method allows for an increase in the number of features analysed; the introduction of a number of geometric morphometry parameters into the measurement protocol; and an increase in the multiplicity of measurements.
The most important feature of this method is the ability to obtain accurate diagnostic data without damaging the plants. We consider it possible to extend the use of the developed method to further field studies of Lycopodiaceae s.l.
Acknowledgements. The authors are deeply grateful to Y.A. Ivanenko for important comments and support, advice, help in identifying plants and obtaining sources of information. A heartfelt appreciation to Y. M. Zinina, who helped to proofread the English version of the text.
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